Compositions and methods for treating infections using cationic peptides alone or in combination with antibiotics

ABSTRACT

Compositions and methods for treating infections, especially bacterial infections, are provided. Indolicidin peptide analogues containing at least two basic amino acids are prepared. The analogues are administered as modified peptides, preferably containing photo-oxidized solubilizer.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of allowed U.S. patentapplication Ser. No. 10/277,233 filed Oct. 18, 2002, which applicationclaims priority from U.S. patent application Ser. No. 09/030,619, filedFeb. 25, 1998 and issued as U.S. Pat. No. 6,503,881, which applicationclaims priority from U.S. Provisional Application No. 60/040,649, filedMar. 10, 1997, and U.S. Provisional Application No. 60/060,099, filedSep. 26, 1997, and U.S. patent application Ser. No. 09/030,619 is acontinuation-in-part of U.S. application Ser. No. 08/915,314, filed Aug.20, 1997, issued as U.S. Pat. No. 6,180,604 on Jan. 30, 2001 whichclaims priority from U.S. Provisional Application No. 60/024,754, filedAug. 21, 1996, and U.S. Provisional Application No. 60/034,949, filedJan. 13, 1997, all of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to methods of treatingmicroorganism-caused infections using cationic peptides or a combinationof cationic peptides and antibiotic agents, and more particularly tousing these peptides and antibiotic agents to overcome acquiredresistance, tolerance, and inherent resistance of an infective organismto the antibiotic agent.

BACKGROUND OF THE INVENTION

For most healthy individuals, infections are irritating, but notgenerally life-threatening. Many infections are successfully combated bythe immune system of the individual. Treatment is an adjunct and isgenerally readily available in developed countries. However, infectiousdiseases are a serious concern in developing countries and inimmunocompromised individuals.

In developing countries, the lack of adequate sanitation and consequentpoor hygiene provide an environment that fosters bacterial, parasitic,fungal and viral infections. Poor hygiene and nutritional deficienciesmay diminish the effectiveness of natural barriers, such as skin andmucous membranes, to invasion by infectious agents or the ability of theimmune system to clear the agents. As well, a constant onslaught ofpathogens may stress the immune system defenses of antibody productionand phagocytic cells (e.g., polymorphic neutrophils) to subnormallevels. A breakdown of host defenses can also occur due to conditionssuch as circulatory disturbances, mechanical obstruction, fatigue,smoking, excessive drinking, genetic defects, AIDS, bone marrowtransplant, cancer, and diabetes. An increasingly prevalent problem inthe world is opportunistic infections in individuals who are HIVpositive.

Although vaccines may be available to protect against some of theseorganisms, vaccinations are not always feasible, due to factors such asinadequate delivery mechanisms and economic poverty, or effective, dueto factors such as delivery too late in the infection, inability of thepatient to mount an immune response to the vaccine, or evolution of thepathogen. For other pathogenic agents, no vaccines are available. Whenprotection against infection is not possible, treatment of infection isgenerally pursued. The major weapon in the arsenal of treatments isantibiotics. While antibiotics have proved effective against manybacteria and thus saved countless lives, they are not a panacea. Theoveruse of antibiotics in certain situations has promoted the spread ofresistant bacterial strains. And of great importance, antibacterials areuseless against viral infections.

A variety of organisms make cationic (positively charged) peptides,molecules used as part of a non-specific defense mechanism againstmicroorganisms. When isolated, these peptides are toxic to a widevariety of microorganisms, including bacteria, fungi, and certainenveloped viruses. One cationic peptide found in neutrophils isindolicidin. While indolicidin acts against many pathogens, notableexceptions and varying degrees of toxicity exist.

Although cationic peptides show efficacy in vitro against a variety ofpathogenic cells including gram-positive bacteria, gram-negativebacteria, and fungi, these peptides are generally toxic to mammals wheninjected, and therapeutic indices are usually quite small. Approaches toreducing toxicity have included development of a derivative or deliverysystem that masks structural elements involved in the toxic response orthat improves the efficacy at lower doses. Other approaches underevaluation include liposomes and micellular systems to improve theclinical effects of peptides, proteins, and hydrophobic drugs, andcyclodextrins to sequester hydrophobic surfaces during administration inaqueous media. For example, attachment of polyethylene glycol (PEG)polymers, most often by modification of amino groups, improves themedicinal value of some proteins such as asparaginase and adenosinedeaminase, and increases circulatory half-lives of peptides such asinterleukins.

None of these approaches are shown to improve administration of cationicpeptides. For example, methods for the stepwise synthesis of polysorbatederivatives that can modify peptides by acylation reactions have beendeveloped, but acylation alters the charge of a modified cationicpeptide and frequently reduces or eliminates the antimicrobial activityof the compound. Thus, for delivery of cationic peptides, as well asother peptides and proteins, there is a need for a system combining theproperties of increased circulatory half-lives with the ability to forma micellular structure.

The present invention discloses analogues of indolicidin, designed tobroaden its range and effectiveness, and further provide other relatedadvantages. The present invention also provides methods and compositionsfor modifying peptides, proteins, antibiotics and the like to reducetoxicity, as well as providing other advantages.

In addition neither antibiotic therapy alone of cationic peptide therapyalone can effectively combat all infections. By expanding the categoriesof microorganisms that respond to therapy, or by overcoming theresistance of a microorganism to antibiotic agents, health and welfarewill be improved. Additionally quality of life will be improved, due to,for example, decreased duration of therapy, reduced hospital stayincluding high-care facilities, with the concomitant reduced risk ofserious nosocomial (hospital-acquired) infections.

The present invention discloses cationic peptides, including analoguesof indolicidin, cecropin/melittin fusion peptides, in combination withantibiotics such that the combination either synergistic, able toovercome microorganismal tolerance, able to overcome resistance toantibiotic treatment, or further provides other related advantages.

BRIEF SUMMARY OF THE INVENTION

The present invention generally provides the co-administration ofcationic peptides with an antibiotic agent and also provides indolicidinanalogues.

In related aspects, an indolicidin analogue is provided, comprising upto 25 amino acids and containing the formula: RXZXXZXB (SEQ ID NO:1);BXZXXZXB (SEQ ID NO:2) wherein at least one Z is valine; BBBXZXXZXB (SEQID NO:3); BXZXXZXBBB_(n)(AA)_(n)MILBBAGS (SEQ ID NOs:5-8);BXZXXZXBB(AA)_(n)M (SEQ ID NOs:9-10); LBB_(n)XZ_(n)XXZ_(n)XRK (SEQ IDNOs:11-18); LK_(n)XZXXZXRRK (SEQ ID NOs:19-20); BBXZXXZXBBB (SEQ IDNO:21), wherein at least two X residues are phenylalanine; BBXZXXZXBBB(SEQ ID NO:22), wherein at least two X residues are tyrosine; andwherein Z is proline or valine; X is a hydrophobic residue; B is a basicamino acid; AA is any amino acid, and n is 0 or 1. In preferredembodiments, Z is proline, X is tryptophan and B is arginine or lysine.In other aspects, indolicidin analogues having specific sequences areprovided. In certain embodiments, the indolicidin analogues are coupledto form a branched peptide. In other embodiments, the analogue has oneor more amino acids altered to a corresponding D-amino acid, and incertain preferred embodiments, the N-terminal and/or the C-terminalamino acid is a D-amino acid. Other preferred modifications includeanalogues that are acetylated at the N-terminal amino acid, amidated atthe C-terminal amino acid, esterified at the C-terminal amino acid,modified by incorporation of homoserine/homoserine lactone at theC-terminal amino acid, and conjugated with polyethylene glycol orderivatives thereof.

In other aspects, the invention provides an isolated nucleic acidmolecule whose sequence comprises one or more coding sequences of theindolicidin analogues, expression vectors, and host cells transfected ortransformed with the expression vector.

Other aspects provide a pharmaceutical composition comprising at leastone indolicidin analogue and a physiologically acceptable buffer,optionally comprising an antibiotic agent.

In other embodiments, the pharmaceutical composition further comprisesan antiviral agent, an antiparasitic agent; and an antifungal agent. Inyet other embodiments, the composition is incorporated in a liposome ora slow-release vehicle.

In yet another aspect, the invention provides a method of treating aninfection, comprising administering to a patient a therapeuticallyeffective amount of a pharmaceutical composition. The infection may becaused by, for example, a microorganism, such as a bacterium (e.g.,Gram-negative or Gram-positive bacterium or anaerobe; parasite orvirus).

In other aspects, a composition is provided, comprising an indolicidinanalogue and an antibiotic. In addition, a device, which may be amedical device, is provided that is coated with the indolicidin analogueand may further comprise an antibiotic agent.

In other aspects, antibodies that react specifically with any one of theanalogues described herein are provided. The antibody is preferably amonoclonal antibody or single chain antibody.

In a preferred aspect, the invention provides a composition comprising acompound modified by derivatization of an amino group with a conjugatecomprising activated polyoxyalkylene and a lipophilic moiety. Inpreferred embodiments, the conjugate comprises sorbitan linkingpolyoxyalkylene glycol and fatty acid, and more preferably ispolysorbate. In preferred embodiments, the fatty acid is from 12-18carbons, and the polyoxyalkylene glycol is polyoxyethylene, such as witha chain length of from 2 to 100. In certain embodiments, the compound isa peptide or protein, such as a cationic peptide (e.g., indolicidin oran indolicidin analogue). In preferred embodiments, the polyoxyalkyleneglycol is activated by irradiation with ultraviolet light or bytreatment with ammonium persulfate.

The invention also provides a method of making a compound modified witha conjugate of an activated polyoxyalkylene and a lipophilic moiety,comprising: (a) freezing a mixture of the conjugate of an activatedpolyoxyalkylene and lipophilic moiety with the compound; and (b)lyophilizing the frozen mixture; wherein the compound has a free aminogroup. In preferred embodiments, the compound is a peptide orantibiotic. In other preferred embodiments, the mixture in step (a) isin an acetate buffer. In a related aspect, the method comprises mixingthe conjugate of an activated polyoxyalkylene and lipophilic moiety withthe compound; for a time sufficient to form modified compounds, whereinthe mixture is in a carbonate buffer having a pH greater than 8.5 andthe compound has a free amino group. The modified compound may beisolated by reversed-phase HPLC and/or precipitation from an organicsolvent.

The invention also provides a pharmaceutical composition comprising atleast one modified compound and a physiologically acceptable buffer, andin certain embodiments, further comprises an antibiotic agent, antiviralagent, an antiparasitic agent, and/or antifungal agent. The compositionmay be used to treat an infection, such as those caused by amicroorganism (e.g., bacterium, fungus, parasite and virus).

This invention also generally provides methods for treating infectionscaused by a microorganism using a combination of cationic peptides andantibiotic agents. In one aspect, the method comprises administering toa patient a therapeutically effective dose of a combination of anantibiotic agent and a cationic peptide, wherein administration of anantibiotic agent alone is ineffective. Preferred peptides are provided.

In another aspect, a method of enhancing the activity of an antibioticagent against an infection in a patient caused by a microorganism isprovided, comprising administering to the patient a therapeuticallyeffective dose of the antibiotic agent and a cationic peptide. In yetanother aspect, a method is provided for enhancing the antibioticactivity of lysozyme or nisin, comprising administering lysozyme ornisin with a cationic peptide.

In other aspects, methods of treating an infection in a patient causedby a bacteria that is tolerant to an antibiotic agent, caused by amicroorganism that is inherently resistant to an antibiotic agent; orcaused by a microorganism that has acquired resistance to an antibioticagent; comprises administering to the patient a therapeuticallyeffective dose of the antibiotic agent and a cationic peptide, therebyovercoming tolerance, inherent or acquired resistance to the antibioticagent.

In yet other related aspects, methods are provided for killing amicroorganism that is tolerant, inherently resistant, or has acquiredresistance to an antibiotic agent, comprising contacting themicroorganism with the antibiotic agent and a cationic peptide, therebyovercoming tolerance, inherent resistance or acquired resistance to theantibiotic agent.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth below whichdescribe in more detail certain procedures or compositions (e.g.,plasmids, etc.), and are therefore incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an SDS-PAGE showing the extraction profile of inclusion bodies(ib) from whole cells containing MBI-11 fusion protein. The fusionprotein band is indicated by the arrow head. Lane 1, protein standards;lane 2, total lysate of XL1 Blue without plasmid; lane 3, total lysateof XL1 Blue (pR2h-11, pGP1-2), cultivated at 30° C.; lane 4, totallysate of XL1 Blue (pR2h-11, pGP1-2), induced at 42° C.; lane 5,insoluble fraction of inclusion bodies after Triton X100 wash; lane 6,organic extract of MBI-11 fusion protein; lane 7, concentrated materialnot soluble in organic extraction solvent.

FIG. 2 is an SDS-PAGE showing the expression profile of the MBI-11fusion protein using plasmid pPDR2h-11. Lane 1, protein standards; lane2, organic solvent extracted MBI-11; lane 3, total lysate of XL1 Blue(pPDR2h-11, pGP1-2), cultured at 30° C.; lane 4, total lysate of XL1Blue (pPDR2h-11, pGP1-2), induced at 42° C.

FIGS. 3A-E present time kill assay results for MBI 11CN, MBI 11F4CN, MBI11B7CN, MBI 11F4CN, and MBI 26 plus vancomycin. The number of colonyforming units×10⁻⁴ is plotted versus time.

FIG. 4 is a graph presenting the extent of solubility of MBI 11CNpeptide in various buffers.

FIGS. 5A-B are a reversed phase HPLC profile of MBI 11CN in formulationC1 (5A) and formulation D (5B).

FIG. 6 presents CD spectra of MBI 11CN and MBI 11B7CN.

FIG. 7 presents results of ANTS/DPX dye release of egg PC liposomes atvarious ratios of lipid to protein.

FIG. 8 presents graphs showing the activity of MBI 11B7CN againstmid-log cells grown in terrific broth (TB) or Luria-Bretani broth (LB).

FIG. 9 shows results of treatment of bacteria with MBI 10CN, MBI 11CN,or a control peptide alone or in combination with valinomycin.

FIG. 10 is a graph showing treatment of bacteria with MBI 11B7CN in thepresence of NaCl or Mg²⁺.

FIG. 11 is a graph presenting the in vitro amount of free MBI 11CN inplasma over time. Data is shown for peptide in formulation C1 andformulation D.

FIG. 12 is a graph showing the stability of MBI-11B7CN-cl inheat-inactivated rabbit serum.

FIG. 13 presents HPLC tracings showing the effects of amastatin andbestatin on peptide degradation.

FIG. 14 is a chromatogram showing extraction of peptides in rabbitplasma.

FIG. 15 is a graph presenting change in in vivo MBI 11CN levels in bloodat various times after intravenous injection.

FIG. 16 is a graph presenting change in in vivo MBI 11CN levels inplasma at various times after intraperitoneal injection.

FIG. 17 is a graph showing the number of animals surviving an MSSAinfection after intraperitoneal injection of MBI 10CN, ampicillin, orvehicle.

FIG. 18 is a graph showing the number of animals surviving an MSSAinfection after intraperitoneal injection of MBI 11CN, ampicillin, orvehicle.

FIG. 19 is a graph showing the results of in vivo testing of MBI-11A1CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 20 is a graph showing the results of in vivo testing of MBI-11E3CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 21 is a graph showing the results of in vivo testing of: MBI-11F3CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 22 is a graph showing the results of in vivo testing of MBI-11G2CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 23 is a graph showing the results of in vivo testing of MBI-11CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 24 is a graph showing the results of in vivo testing of MBI-11B1CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 25 is a graph showing the results of in vivo testing of MBI-11B7CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 26 is a graph showing the results of in vivo testing of MBI-11B8CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 27 is a graph showing the results of in vivo testing of MBI-11G4CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 28 displays graphs showing the number of animals surviving an S.epidermidis infection after intravenous injection of MBI 10CN,gentamicin, or vehicle. Panel a, i.v. injection 15 min post-infection;panel b, i.v. injection 60 min post-infection.

FIG. 29 is a graph showing the number of animals surviving an MRSAinfection mice after intravenous injection of MBI 11CN, gentamicin, orvehicle.

FIGS. 30A-30C present RP-HPLC traces analyzing samples for APS-peptideformation after treatment of activated polysorbate with a reducingagent. APS-MBI-11CN peptides are formed via lyophilization in 200 mMacetic acid-NaOH, pH 4.6, 1 mg/ml MBI 11CN, and 0.5% activatedpolysorbate 80. The stock solution of activated 2.0% polysorbate istreated with (a) no reducing agent, (b) 150 mM 2-mercaptoethanol, or (c)150 mM sodium borohydride for 1 hour immediately before use.

FIG. 31 presents RP-HPLC traces monitoring the formation of APS-MBI 11CNover time in aqueous solution. The reaction occurs in 200 mM sodiumcarbonate buffer pH 10.0, 1 mg/ml MBI 11CN, 0.5% activated polysorbate80. Aliquots are removed from the reaction vessel at the indicated timepoints and immediately analyzed by RP-HPLC.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms that areused herein.

The amino acid designations herein are set forth as either the standardone- or three-letter code. A capital letter indicates an L-form aminoacid; a small letter indicates a D-form amino acid.

As used herein, an “antibiotic agent” refers to a molecule that tends toprevent, inhibit, or destroy life. The term “antimicrobial agent” refersto an antibiotic agent specifically directed to a microorganism.

As used herein, “cationic peptide” refers to a peptide that has a netpositive charge within the pH range of 4-10. A cationic peptide is atleast 5 amino acids in length and has at least one basic amino acid(e.g., arginine, lysine, histidine). Preferably, the peptide hasmeasurable anti-microbial activity when administered alone.

As used herein, “indolicidin” refers to an antimicrobial cationicpeptide. Indolicidins may be isolated from a variety of organisms. Oneindolicidin is isolated from bovine neutrophils and is a 13 amino acidpeptide amidated at the carboxy-terminus in its native form (Selsted etal., J. Biol. Chem. 267:4292, 1992). An amino acid sequence ofindolicidin is presented in SEQ ID NO: 1.

As used herein, a “peptide analogue”, “analogue”, or “variant” of acationic peptide, such as indolicidin, is at least 5 amino acids inlength, has at least one basic amino acid (e.g., arginine and lysine)and has anti-microbial activity. Unless otherwise indicated, a namedamino acid refers to the L-form. Basic amino acids include arginine,lysine, histidine and derivatives. Hydrophobic residues includetryptophan, phenylalanine, isoleucine, leucine, valine, and derivatives.

Also included within the scope of the present invention are amino acidderivatives that have been altered by chemical means, such asmethylation (e.g., a methylvaline), amidation, especially of theC-terminal amino acid by an alkylamine (e.g., ethylamine, ethanolamine,and ethylene diamine) and alteration of an amino acid side chain, suchas acylation of the 2-amino group of lysine. Other amino acids that maybe incorporated in the analogue include any of the D-amino acidscorresponding to the 20 L-amino acids commonly found in proteins, iminoamino acids, rare amino acids, such as hydroxylysine, or non-proteinamino acids, such as homoserine and ornithine. A peptide analogue mayhave none or one or more of these derivatives, and D-amino acids. Inaddition, a peptide may also be synthesized as a retro-, inverto- orretro-inverto-peptide.

As used herein “inherent resistance” of a microorganism to an antibioticagent refers to a natural resistance to the action of the agent even inthe absence of prior exposure to the agent. (R. C. Moellering Jr.,Principles of Anti-infective Therapy; In: Principles and Practice ofInfectious Diseases, 4^(th) Edition, Eds.; G. L. Mandell, J. E. Bennett,R. Dolin. Churchill Livingstone, New York USA, 1995, page 200).

As used herein, “acquired resistance” of a microorganism to anantibiotic agent refers to a resistance that is not inhibited by thenormal achievable serum concentrations of a recommended antibiotic agentbased on the recommended dosage. (NCCLS guidelines).

As used herein, “tolerance” of a microorganism to an antibiotic agentrefers to when there is microstatic, rather than microcidal effect ofthe agent. Tolerance is measured by an MBC:MIC ratio greater than orequal to 32. (Textbook of Diagnostic Microbiology, Eds., C. R. Mahon andG. Manuselis, W.B. Saunders Co., Toronto Canada, 1995, page 92).

As noted above, this invention provides methods of treating infectionscaused by a microorganism, methods of killing a microorganism, andmethods of enhancing the activity of an antibiotic agent. In particular,these methods are especially applicable when a microorganism isresistant to an antibiotic agent, by a mechanism, such as tolerance,inherent resistance, or acquired resistance. In this invention,infections are treated by administering a therapeutically effective doseof a cationic peptide alone or in combination with an antibiotic agentto a patient with an infection. Similarly, the combination can becontacted with a microorganism to effect killing.

I. Cationic Peptides

As noted above, a cationic peptide is a peptide that has a net positivecharge within the pH range 4-10. A peptide is at least 5 amino acidslong and preferably not more than 25, 27, 30, 35, or 40 amino acids.Peptides from 12 to 30 residues are preferred. Examples of nativecationic peptides include, but are not limited to, representativepeptides presented in the following table.

TABLE 1 Cationic Peptides Acces- sion Group Name Peptide Origin SequenceNumber Reference* Abaecins Abaecin Honey beeYVPLPNVPQPGRRPFPTFPGQGPFNPKI P15450 Casteels P.  (Apis mellifera) KWPQGYet al., (1990) (SEQ ID NO: 156) Andropins Andropin Fruit flyVFIDILDKVENAIHNAAQVGIGFAKPFEKLI P21663 Samakovlis, C.  (Drosophilia NPKet al., (1991) melanogaster) (SEQ ID NO: 157) Apidaecins Apidaecin IALymph fluid of  GNNRPVYIPQPRPPHPRI P11525 Casteels, P.  honey bee(SEQ ID NO: 158) et al., (1989) (Apis mellifera) Apidaecin IBLymph fluid of  GNNRPVYIPQPRPPHPRL P11526 Casteels, P.  honey bee(SEQ ID NO: 159) et al., (1989) (Apis mellifera) Apidaecin IILymph fluid of  GNNRPIYIPQPRPPHPRL P11527 Casteels, P.  honey bee(SEQ ID NO: 160) et al., (1989) (Apis mellifera) AS AS-48 Streptococcus 7.4 kDa Galvez, A.,  faecalis subsp.  et al., (1989) Liquefacines S-48Bactenecins Bactenecin Cytoplasmic   RLCRIVVIRVCR A33799 Romeo, D. granules (SEQ ID NO: 161) et al., (1988) of bovine neutrophils Bac Bac5Cytoplasmic   RFRPPIRRPPIRPPFYPPFRPPIRPPIFPPI B36589 Frank, R. W. granules  RPPFRPPLRFP et al., (1990) of bovine (SEQ ID NO: 162)neutrophils Bac7 Cytoplasmic   RRIRPRPPRLPRPRPRPLPFPRPGPRPIP A36589Frank, R. W.  granules RPLPFPRPGPRPIPRPLPFPRPGPRPIPR et al., (1990)of bovine  P neutrophils (SEQ ID NO: 163) Bactericidins Bactericidin Tobacco hornworm WNPFKELERAGQRVRDAVISAAPAVATV P14662 Dickinson, L.  B2larvae hemolymph GQAAAIARG* et al., (1988) (Manduca sexta)(SEQ ID NO: 164) Bactericidin  Tobacco hornwormWNPFKELERAGQRVRDAIISAGPAVATV P14663 Dickinson, L. B-3 larvae hemolymphGQAAAIARG et al., (1988) (Manduca sexta) (SEQ ID NO: 165) Bactericidin Tobacco hornworm WNPFKELERAGQRVRDAIISAAPAVATV P14664 Dickinson, L.  B-4larvae hemolymph GQAAAIARG* et al., (1988) (Manduca sexta)(SEQ ID NO: 166) Bactericidin  Tobacco hornwormWNPFKELERAGQRVRDAVISAAAVATVG P14665 Dickinson, L.,  B-5Plarvae hemolymph QAAAIARGG* et al., (1988) (Manduca sexta)(SEQ ID NO: 167) Bacteriocins Bacteriocin Streptococcus 4.8 kDaTakada, K.,  C3603 mutants et al., (1984) Bacteriocin Staphylococcus 5 kDa Nakamura, T.,  IY52 aureus et al., (1983) Bombinins BombininYellow-bellied GIGALSAKGALKGLAKGLAZHFAN* P01505 Csordas,   toad(SEQ ID NO: 168) A., and  (Bombina Michl, H. variegata) (1970) BLP-1Asian Toad GIGASILSAGKSALKGLAKGLAEHFAN* M76483 Gibson, B. W.  (Bombina (SEQ ID NO: 169) et al., (1991) orientalis) BLP-2 Asian ToadGIGSAILSAGKSALKGLAKGLAEHFAN* B41575 Gibson, B. W.  (Bombina (SEQ ID NO: 170) et al., (1991) orientalis) Bombolitins Bombolitin Bumblebee venom IKITTMLAKLGKVLAHV* P10521 Argiolas,   BI (Megabombus(SEQ ID NO: 171) A. and pennsylvanicus) Pisano, J. J. (1985) Bombolitin Bumblebee venom SKITDILAKLGKVLAHV* P07493 Argiolas,   BII (Megabombus(SEQ ID NO: 172) A. and pennsylvanicus) Pisano, J. J. (1985) BPTI BovineBovine Pancreas RPDFCLEPPYTGPCKARIIRYFYNAKAGL P00974 Creighton, Pancreatic CQTFVYGGCRAKRNNFKSAEDCMRTCG T. and Trypsin GA Charles, I. G. Inhibitor  (SEQ ID NO: 173) (1987) (BPTI) Brevinins Brevinin-1EEuropean frog FLPLLAGLAANFLPKIFCKITRKC S33729 Simmaco, M. (Rana esculenta) (SEQ ID NO: 174) et al., (1993) Brevinin-2EGIMDTLKNLAKTAGKGALQSLLNKASCK S33730 Simmaco, M.  LSGQC et al., (1993)(SEQ ID NO: 175) Cecropins Cecropin A Silk mothKWKLFKKIEKVGQNIRDGIIKAGPAVAVV M63845 Gudmundsson,  (Hyalophora GQATQIAK* G. H. et al.,  cecropia) (SEQ ID NO: 176) (1991) Cecropin BSilk moth KWKVFKKIEKMGRNIRNGIVKAGPAIAVL Z07404 Xanthopoulos,  (Hyalophora  GEAKAL* G. cecropia) (SEQ ID NO: 177) et al.(1988)Cecropin C Fruit fly GWLKKLGKRIERIGQHTRDATIQGLGIAQ Z11167 Tryselius, Y. (Drosophila QAANVAATARG* et al. (1992) melanogaster) (SEQ ID NO: 178)Cecropin D Silk moth pupae WNPFKELEKVGQRVRDAVISAGPAVATV P01510Hultmark, D.  (Hyalophora  AQATALAK* et al., (1982) cecropia)(SEQ ID NO: 179) Cecropin P₁ Pig small  SWLSKTAKKLENSAKKRISEGIAIAIQGGP14661 Lee, J. -Y.   intestine PR et al., (1989) (sus scrofa)(SEQ ID NO: 180) Charybdtoxins Charybdtoxin Scorpion venomZFTNVSCTTSKECWSVCQRLHNTSRGK P13487 Schweitz, H.  (Leiurus guin-CMNKKCRCYS et al., (1989) questriatus  (SEQ ID NO: 181) hebraeus)Coleoptericins Coleoptericin Beetle 8.1 kDa A41711 Bulet, P.  (Zophabas  et al.,  atratus) (1991) Crabolins Crabolin European hornet FLPLILRKIVTAL* A01781 Argiolas,   venom (SEQ ID NO: 182) A. and(Vespa crabo) Pisano, J. J. (1984) Defensins- Cryptdin 1 Mouse intestineLRDLVCYCRSRGCKGRERMNGTCRKG A43279 Selsted, M. E.  alpha (Mus musculus)HLLYTLCCR et al., (1992) (SEQ ID NO: 183) Cryptdin 2 Mouse intestineLRDLVCYCRTRGCKRRERMNGTCRKGH C43279 Selsted, M. E.  (Mus musculus)LMYTLCCR et al., (1992) (SEQ ID NO: 184) MCP1 Rabbit alveolarVVCACRRALCLPRERRAGFCRIRGRIHP M28883 Selsted, M.  macrophages LCCRRet al., (1983) (Oryctolagus  (SEQ ID NO: 185) cuniculus) MCP2Rabbit alveolar VVCACRRALCLPLERRAGFCRIRGRIHPL M28073 Ganz, T.  macrophages CCRR et al., (1989) (Oryctolagus  (SEQ ID NO: 186)cuniculus) GNCP-1 Guinea pig RRCICTTRTCRFPYRRLGTCIFQNRVYTF S21169Yamashita,  (Cavia cutteri) CC T. and (SEQ ID NO: 187) Saito, K., (1989)GNCP-2 Guinea pig RRCICTTRTCRFPYRRLGTCLFQNRVYT X63676 Yamashita, (Cavia cutteri) FCC T, and  (SEQ ID NO: 188) Saito, K., (1989) HNP-1Azurophil granules  ACYCRIPACIAGERRYGTCIYQGRLWAF P11479 Lehrer, R.  of human  CC et al., (1991) neutrophils (SEQ ID NO: 189) HNP-2Azurophil granules  CYCRIPACIAGERRYGTCIYQGRLWAFC P11479 Lehrer, R.  of human  C et al., (1991) neutrophils (SEQ ID NO: 190) NP-1Rabbit neutrophils VVCACRRALCLPRERRAGFCRIRGRIHP P01376 Ganz, T.  (Oryctolagus  LCCRR et al., (1989) cuniculus) (SEQ ID NO: 191) NP-2Rabbit neutrophils VVCACRRALCLPLERRAGFCRIRGRIHPL P01377 Ganz, T.  (Oryctolagus  CCRR et al., (1989) cuniculus) (SEQ ID NO: 192) RatNP-1Rat neutrophils VTCYCRRTRCGFRERLSGACGYRGRIY A60113 Eisenhauer,  (Rattus  RLCCR P. B. et al.,  norvegicus) (SEQ ID NO: 193) (1989)RatNP-2 Rat neutrophils VTCYCRSTRCGFRERLSGACGYRGRIY Eisenhauer,  (Rattus RLCCR P. B. et al.,  norvegicus) (SEQ ID NO: 194) (1989)Defensins- BNBD-1 Bovine neutrophils DFASCHTNGGICLPNRCPGHMIQIGICFR127951 Selsted, M. E.  beta PRVKCCRSW et at., (1993) (SEQ ID NO: 195)BNBD-2 Bovine neutrophils VRNHVTCRINRGFCVPIRCPGRTRQIGT 127952Selsted, M. E.,  CFGPRIKCCRSW et al., (1993) (SEQ ID NO: 196) TAPBovine tracheal  NPVSCVRNKGICVPIRCPGSMKQIGTCV P25068 Diamond, G.  mucosaGRAVKCCRKK et al., (1991) (Bos taurus) (SEQ ID NO: 197) Defensins-Sapecin Flesh fly ATCDLLSGTGINHSACAAHCLLRGNRGG J04053 Hanzawa, H. insect (Sacrophaga  YCNGKAVCVCRN et al., (1990) peregrina)(SEQ ID NO: 198) Insect  Dragonfly larvae GFGCPLDQMQCHRHCQTITGRSGGYCSP80154 Bulet, P.   defensin (Aeschna cyanea) GPLKLTCTCYR et al., (1992)(SEQ ID NO: 199) Defensins- Scorpion ScorpionGFGCPLNQGACHRHCRSIRRRGGYCAG Cociancich, S.  scorpion defensin (LeiurusFFKQTCTCYRN et al., (1993) quinquestriatus) (SEQ ID NO: 200)Dermaseptins Dermaseptin South American ALWKTMLKKLGTMALHAGKAALGAADTIP24302 Mor, A.,   arboreal frog SQTQ et al., (1991) (Phyllomedusa(SEQ ID NO: 201) sauvagii) Diptericins Diptericin Nesting-suckling  9 kDa X15851 Reichhardt,   blowfly J. M. et al.,  (Phormia  (1989)terranovae) Drosocins Drosocin Fruit fly GKPRPYSPRPTSHPRPIRV S35984Bulet, P.   (Drosophila (SEQ ID NO: 202) et al., (1993) melanogaster)Esculentins Esculentin European frog GIFSKLGRKKIKNLLISGLKNVGKEVGMDS33731 Simmaco, M.  (Rana esculenta) VVRTGIDIAGCKIKGEC et al., (1993)(SEQ ID NO: 203) Indolicidins Indolicidin Bovine neutrophilsILPWKWPWWPWRR* A42387 Selsted, M.  (SEQ ID NO: 204) et al., (1992)Lactoferricins Lactoferri-  N terminal region  FKCRRWQWRMKKLGAPSITCVRRAFM63502 Bellamy, W.  cin B of bovine  (SEQ ID NO: 205) et al., lactoferrin (1992b) Lantibiotics Nisin Lactococcus lactisITSISLCTPGCKTGALMGCNMKTATCHC P13068 Hurst, A.  subsp. Lactis SIHVSK(1981) (bacterium) (SEQ ID NO: 206) Pep 5 StaphylococcusTAGPAIRASVKQCQKTLKATRLFTVSCK P19578 Keletta, C.   epidermidis GKNGCKet al., (1989) (SEQ ID NO: 207) Subtilin Bacillus subtilisMSKFDDFDLDVVKVSKQDSKITPQWKSE P10946 Banerjee,  (bacterium)SLCTPGCVTGALQTCFLQTLTCNCKISK S. and Hansen,  (SEQ ID NO: 208)J. N. (1988) Leukocins Leukocin Leuconostoc  KYYGNGVHCTKSGCSVNWGEAFSAGVS65611 Hastings,   A-val 187 gelidum HRLANGGNGFW J. W. UAL 187(SEQ ID NO: 209) et al., (1991) (bacterium) Magainins Magainin IAmphibian skin GIGKFLHSAGKFGKAFVGEIMKS* A29771 Zasloff, M. (Xenopus laevis) (SEQ ID NO: 210) (1987) Magainin II Amphibian skinGIGKFLHSAKKFGKAFVGEIMNS* A29771 Zasloff, M.  (Xenopus laevis)(SEQ ID NO: 211) (1987) PGLa Amphibian skin GMASKAGAIAGKIAKVALKAL*X13388 Kuchler, K.  (Xenopus iaevis) (SEQ ID NO: 212) et al., (1989) PGQAmphibian stomach GVLSNVIGYLKKLGTGALNAVLKQ Moore, K. S. (Xenopus iaevis) (SEQ ID NO: 213) et al., (1989) XPF Amphibian skinGWASKIGQTLGKIAKVGLKELIQPK P07198 Sures, I. And  (Xenopus iaevis)(SEQ ID NO: 214) Crippa, M.  (1984) Mastoparans Mastoparan Wasp venomINLKALAALAKKIL* P01514 Bernheimer,  (Vespula lewisii) (SEQ ID NO: 215)A. and Rudy, B. (1986) Melittins Melittin Bee venomGIGAVLKVLTTGLPALISWIKRKRQQ P01504 Tosteson,  (Apis mellifera)(SEQ ID NO: 216) M. T. and    Tosteson, D. C. (1984) PhormicinsPhormicin A Nestling-suckling ATCDLLSGTGINHSACAAHCLLRGNRGG P10891Lambert, J. blowfly YCNGKGVCVCRN et al., (1989) (Phormia (SEQ ID NO: 217) terranovae) Phormicin B Nestling-sucklingATCDLLSGTGINHSACAAHCLLRGNRGG P10891 Lambert, J.  blowfly YCNRKGVCVRNet al., (1989) (Phormia  (SEQ ID NO: 218) terranovae) PolyphemusinsPolyphemusin  Atlantic horseshoe  RRWCFRVCYRGFCYRKCR* P14215Miyata, T.   I crab (SEQ ID NO: 219) et al., (1989) (Limulus polyphemus) Polyphemusin Atlantic horseshoe  RRWCFRVCYKGFCYRKCR* P14216Miyata, T.   II crab (SEQ ID NO: 220) et al., (1989) (Limulus polyphemus) Protegrins Protegrin  Porcine leukocytes RGGRLCYCRRRFCVCVGRS34585 Kokryakov,   I (sus scrofa) (SEQ ID NO: 221) V. N. et al., (1993)Protegrin  Porcine leukocytes RGGRLCYCRRRFCICV S34586 Kikryakov,   II(sus scrofa) (SEQ ID NO: 222) V. N. et al., (1993) Protegrin Porcine leukocytes RGGGLCYCRRRFCVCVGR S34587 Kokryakov,   III(sus scrofa) (SEQ ID NO: 223) V. N. et al., (1993) Royalisins RoyalisinRoyal Jelly VTCDLLSFKGQVNDSACAANCLSLGKAG P17722 Fujiwara, S. (Apis mellifera) GHCEKGVCICRKTSFKDLWDKYF et al., (1990) (SEQ ID NO: 224)Sarcotoxins Sarcotoxin  Flesh fly GWLKKIGKKIERVGQHTRDATIQGLGIAQ P08375Okada, M. and  IA (Sacrophaga QAANVAATAR* Natori S.,  peregrina)(SEQ ID NO: 225) (1985b) Sarcotoxin  Flesh flyGWLKKIGKKIERVGQHTRDATIQVIGVAQ P08376 Okada, M. and  IB (Sacrophage QAANVAATAR* Natori S.,  peregrina) (SEQ ID NO: 226) (1985b) SeminalSeminal- Bovine seminal SDEKASPDKHHRFSLSRYAKLANRLANP S08184 Reddy, plasmins plasmin plasma KLLETFLSKWIGDRGNRSV E. S. P. and   (Bos taurus)(SEQ ID NO: 227) Bhargava, P. M. (1979) Tachyplesins Tachyplesin Horseshoe crab KWCFRVCYRGICYRRCR* P23684 Nakamura, T.  I (Tachypleus (SEQ ID NO: 228) et al., (1988) tridentatus) Tachyplesin  Horseshoe crabRWCFRVCYRGICYRKCR* P14214 Muta, T.  II (Tachypleus (SEQ ID NO: 229)et al., (1990) tridentatus) Thionins Thionin Barley leafKSCCKDTLARNCYNTCRFAGGSRPVCA S00825 Bohlmann, H. BTH6 (Hordeum vulgare)GACRCKIISGPKCPSDYPK et al., (1988) (SEQ ID NO: 230) Toxins Toxin 1Waglers pit viper  GGKPDLRPCIIPPCHYIPRPKPR P24335 Schmidt, J. J.  venom(SEQ ID NO: 231) et al., (1992) (Trimeresurus  wagleri) Toxin 2Sahara scorpion VKDGYIVDDVNCTYFCGRNAYCNEECTK P01484 Bontems, F., (Androctonus LKGESGYCQWASPYGNACYCKLPDHVR et al., (1991) australisTKGPGRCH Hector) (SEQ ID NO: 232) Argiolas and Pisano, (1984). JBC 259,10106; Argiolas and Pisano, (1985). JBC 260, 1437; Banerjee and Hansen,(1988). JBC 263, 9508; Bellamy et al., (1992). J. Appl. Bacter. 73, 472;Bernheimer and Rudy, (1986). BBA 864, 123; Bohlmann et al., (1988). EMBOJ. 7, 1559; Bontems et al., (1991). Science 254, 1521; Bulet et al.,(1991). JBC 266, 24520; Bulet et al. (1992). Eur. J. Biochem. 209, 977;Bulet et al., (1993). JBC 268, 14893; Casteels et al., (1989). EMBO J.8, 2387; Casteels et al., (1990). Eur. J. Biochem. 187, 381; Cociancichet al., (1993). BBRC 194, 17; Creighton and Charles, (1987). J. Mol.Biol. 194, 11; Csordas and Michl, (1970). Monatsh Chemistry 101, 182;Diamond et al., (1991). PNAS 88, 3952; Dickinson et al., (1988). JBC263, 19424; Eisenhauer et al., (1989). Infect. and Imm. 57, 2021; Franket al., (1990). JBC 265, 18871; Fujiwara et al., (1990). JBC 265, 11333;Gálvez et al., (1989). Antimicrobial Agents and Chemotherapy 33, 437;Ganz et al., (1989). J. Immunol. 143, 1358; Gibson et al., (1991). JBC266, 23103; Gudmundsson et al., (1991). JBC 266, 11510; Hanzawa et al.,(1990). FEBS Letters 269, 413; Hastings et al., (1991). J. ofBacteriology 173, 7491; Hultmark et al., (1982). Eur. J. Biochem. 127,207; Hurst, A. (1981). Adv. Appl. Micro. 27, 85; Kaletta et al., (1989).Archives of Microbiology 152, 16; Kokryakov et al., (1993). FEBS Letters327, 231; Kuchler et al., (1989) Eur. J. Biochem. 179, 281; Lambert etal., (1989). PNAS 86, 262; Lee et al., (1989). PNAS 86, 9159; Lehrer atal., (1991). Cell 64, 229; Miyata et al., (1989). J. of Biochem. 106,663; Moore et al., (1991). JBC 266, 19851; Mor et al., (1991).Biochemistry 30, 8824; Muta et al., (1990). J. Biochem. 108, 261;Nakamura at al., (1988). JBC 263, 16709; Nakamura et al., (1983).Infection and Immunity 39, 609; Okada and Natori (1985). Biochem. J.229, 453; Reddy and Bhargava, (1979). Nature 279, 725; Reichhart et al.,(1989). Eur. J. Biochem. 182, 423; Romeo et al., (1988). JBC 263, 9573;Samakovlis et al., (1991). EMBO J. 10, 163; Schmidt et al., (1992).Toxicon 30, 1027; Schweitz et al., (1989). Biochem. 28, 9708; Selsted etal., (1983). JBC 258, 14485; Selsted et al., (1992). JBC 267, 4292;Simmaco et al., (1993). FEBS Letters 324, 159; Sures and Crippa (1984).PNAS 81, 380; Takada et al., (1984). Infect. and Imm. 44, 370; Tostesonand Tosteson, (1984). Biophysical J. 45, 112; Tryselius et al., (1992).Eur. J. Biochem. 204, 395; Xanthopoulos et al., (1988). Eur. J. Biochem.172, 371; Yamashita and Saito, (1989). Infect. and Imm. 57, 2405;Zasloff, M. (1987). PNAS 84, 5449.

In addition to the peptides listed above, chimeras and analogues ofthese peptides are useful within the context of the present invention.For this invention, analogues of native cationic peptides must retain anet positive charge, but may contain D-amino acids, amino acidderivatives, insertions, deletions, and the like, some of which arediscussed below. Chimeras include fusions of cationic peptide, such asthe peptides of fragments thereof listed above, and fusions of cationicpeptides with non-cationic peptides.

As described herein, modification of any of the residues including theN- or C-terminus is within the scope of the invention. A preferredmodification of the C-terminus is amidation. Other modifications of theC-terminus include esterification and lactone formation. N-terminalmodifications include acetylation, acylation, alkylation, PEGylation,myristylation, and the like. Additionally, the peptide may be modifiedto form an polymer-modified peptide as described below. The peptides mayalso be labeled, such as with a radioactive label, a fluorescent label,a mass spectrometry tag, biotin and the like.

Unless otherwise indicated, a named amino acid refers to the L-form.Basic amino acids include arginine, lysine, histidine, and derivatives.Hydrophobic residues include tryptophan, phenylalanine, isoleucine,leucine, valine, and derivatives. The peptide may contain derivatives ofamino acids that have been altered by chemical means, such asmethylation (e.g., α-methylvaline), amidation, especially of theC-terminal amino acid by an alkylamine (e.g., ethylamine, ethanolamine,and ethylene diamine) and alteration of an amino acid side chain, suchas acylation of the ε-amino group of lysine. Other amino acids that maybe incorporated include any of the D-amino acids corresponding to the 20L-amino acids commonly found in proteins, rare amino acids, such ashydroxylysine, or non-protein amino acids, such as homoserine andornithine. A peptide may have none or one or more of these derivatives,and may contain D-amino acids (specified as a lower case letter whenusing the 1-letter code). Furthermore, modification of the N- orC-terminus is within the scope of the invention. A preferredmodification of the C-terminus is amidation. Other modifications of theC-terminus include ester additions. N-terminal modifications includeacetylation, myristlyation, and the like.

A. Indolicidin and Analogues

As noted above, the present invention provides cationic peptides,including indolicidin and indolicidin analogues. Analogues includepeptides that have one or more insertions, deletions, modified aminoacids, D-amino acids and the like. These analogues may be synthesized bychemical methods, especially using an automated peptide synthesizer, orproduced by recombinant methods. The choice of an amino acid sequence isguided by a general formula presented herein.

The indolicidin analogues of the present invention are at least 5 or 7amino acids in length and preferably not more than 15, 20, 25, 27, 30,or 35 amino acids. Analogues from 9 to 14 residues are preferred.General formulas for peptide analogues in the scope of the presentinvention may be set forth as:

RXZXXZXB (SEQ ID NO: 1)  (1) BXZXXZXB (SEQ ID NO: 2)  (2) BBBXZXXZXB(SEQ ID NO: 3)  (3) BXZXXZXBBB_(n)(AA)_(n)MILBBAGS (SEQ ID NOs: 5-8) (4) BXZXXZXBB(AA)_(n)M (SEQ ID NOs: 9-10)  (5) LBB_(n)XZ_(n)XXZ_(n)XRK(SEQ ID NOs: 11-18)  (6) LK_(n)XZXXZXRRK (SEQ ID NOs: 19-20)  (7)BBXZXXZXBBB (SEQ ID NO: 21)  (8) BBXZXXZXBBB (SEQ ID NO: 22)  (9)BXXBZBXBXZB (SEQ ID NO: 4) (10)

wherein standard single letter amino abbreviations are used and; Z isproline, glycine or a hydrophobic residue, and preferably Z is prolineor valine; X is a hydrophobic residue, such as tryptophan,phenylalanine, isoleucine, leucine and valine, and preferablytryptophan; B is a basic amino acid, preferably arginine or lysine; AAis any amino acid, and n is 0 or 1. In formula (2), at least one Z isvaline; in formula (8), at least two Xs are phenylalanine; and informula (9), at least two Xs are tyrosine. Additional residues may bepresent at the N-terminus, C-terminus, or both.

B. Cecropin Peptides

Cecropins are cationic peptides that have antimicrobial activity againstboth Gram-positive and Gram-negative bacteria. Cecropins have beenisolated from both invertebrates (e.g., insect hemolymph) as well asvertebrates (e.g. pig intestines). Generally, these peptides are 35 to39 residues. An exemplary cecropin has the sequenceKWKLFKKIEKVGQNIRDGIIKAGPAVAWGQATQIAK (SEQ ID NO:176). Some additionalcecropin sequences are presented in Table 1. Within the context of thisinvention, cecropins include analogues that have one or more insertions,deletions, modified amino acids, D-amino acids and the like.

C. Melittin Peptides

Melittin is a cationic peptide found in bee venom. An amino acidsequence of an exemplary melittin peptide is GIGAVLKVLTTGLPALISWIKRKKRQQ(SEQ ID NO:216). Like the cecropins, melittin exhibits antimicrobialactivity against both Gram-positive and Gram-negative bacteria. Withinthe context of this invention, melittin includes analogues that have oneor more insertions, deletions, modified amino acids, D-amino acids andthe like.

D. Cecropin-Melittin Chimeric Peptides

As noted herein, cationic peptides include fusion peptides of nativecationic peptides and analogues of fusion peptides. In particular,fusions of cecropin and melittin are provided. An exemplary fusion hasthe sequence: cecropin A (residues 1-8)/melittin (residues 1-18). Otherfusion peptides useful within the context of this invention aredescribed by the general formulas below.

(SEQ ID NO: 128) K W K R₂ R₁ R₁ R₂ R₂ R₁ R₂ R₂ R₁ R₁ R₂ R₂ V  L T T G L P A L I S (SEQ ID NO: 129)K W K R₂ R₁ R₁ R₂ R₂ R₁ R₂ R₂ R₁ R₁ R₂ R₂ V   V T T A K P L I S S(SEQ ID NO: 130) K W K R₂ R₁ R₁ R₂ R₂ R₁ R₂ R₂ R₁ R₁ R₂ R₂ I  L T T G L P A L I S (SEQ ID NO: 131)K W K R₂ R₁ R₁ R₂ R₂ R₁ R₂ R₂ R₁ R₁ R₂ R₂ G   G L L S N I V T S L(SEQ ID NO: 132) K W K R₂ R₁ R₁ R₂ R₂ R₁ R₂ R₂ R₁ R₁ R₂ R₂ G  P I L A N L V S I V (SEQ ID NO: 133)K K W W R R R₁ R₁ R₂ R₁ R₁ R₂ R₂ G P A L S N V (SEQ ID NO: 134-144)K K W W R R X (SEQ ID NO: 145-155) K K W W K Xwherein R₁ is a hydrophobic amino acid residue, R₂ is a hydrophilicamino acid residue, and X is from about 14 to 24 amino acid residues.

E. Drosocin and Analogues

As noted herein, cationic peptides include drosocin and drosocinanalogues. Drosocins are isolated from Drosophila melanogaster. Anexemplary drosocin is a 19 amino acid peptide having the sequence:GKPRPYSPRPTSHPRPIRV (SEQ ID NO:202); GenBank Accession No. S35984).Analogues of drosocin include peptides that have insertions, deletions,modified amino acids, D-amino acids and the like.

F. Peptide Synthesis

Peptides may be synthesized by standard chemical methods, includingsynthesis by automated procedure. In general, peptide analogues aresynthesized based on the standard solid-phase Fmoc protection strategywith HATU as the coupling agent. The peptide is cleaved from thesolid-phase resin with trifluoroacetic acid containing appropriatescavengers, which also deprotects side chain functional groups. Crudepeptide is further purified using preparative reversed-phasechromatography. Other purification methods, such as partitionchromatography, gel filtration, gel electrophoresis, or ion-exchangechromatography may be used.

Other synthesis techniques, known in the art, such as the tBocprotection strategy, or use of different coupling reagents or the likecan be employed to produce equivalent peptides.

Peptides may be synthesized as a linear molecule or as branchedmolecules. Branched peptides typically contain a core peptide thatprovides a number of attachment points for additional peptides. Lysineis most commonly used for the core peptide because it has one carboxylfunctional group and two (alpha and epsilon) amine functional groups.Other diamino acids can also be used. Preferably, either two or threelevels of geometrically branched lysines are used; these cores form atetrameric and octameric core structure, respectively (Tam, Proc. Natl.Acad. Sci. USA 85:5409, 1988). Schematically, examples of these coresare represented as shown (SEQ ID NO:92):

The attachment points for the peptides are typically at their carboxylfunctional group to either the alpha or epsilon amine groups of thelysines. To synthesize these multimeric peptides, the solid phase resinis derivatized with the core matrix, and subsequent synthesis andcleavage from the resin follows standard procedures. The multimericpeptides may be used within the context of this invention as for any ofthe linear peptides and are preferred for use in generating antibodiesto the peptides.

G. Recombinant Production of Peptides

Peptides may alternatively be synthesized by recombinant production (seee.g., U.S. Pat. No. 5,593,866). A variety of host systems are suitablefor production of the peptide analogues, including bacteria (e.g., E.coli), yeast (e.g., Saccharomyces cerevisiae), insect (e.g., Sf9), andmammalian cells (e.g., CHO, COS-7). Many expression vectors have beendeveloped and are available for each of these hosts. Generally, bacteriacells and vectors that are functional in bacteria are used in thisinvention. However, at times, it may be preferable to have vectors thatare functional in other hosts. Vectors and procedures for cloning andexpression in E. coli are discussed herein and, for example, in Sambrooket al. (Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1987) and in Ausubel et al.(Current Protocols in Molecular Biology, Greene Publishing Co., 1995).

A DNA sequence encoding a cationic peptide is introduced into anexpression vector appropriate for the host. In preferred embodiments,the gene is cloned into a vector to create a fusion protein. The fusionpartner is chosen to contain an anionic region, such that a bacterialhost is protected from the toxic effect of the peptide. This protectiveregion effectively neutralizes the antimicrobial effects of the peptideand also may prevent peptide degradation by host proteases. The fusionpartner (carrier protein) of the invention may further function totransport the fusion peptide to inclusion bodies, the periplasm, theouter membrane, or the extracellular environment. Carrier proteinssuitable in the context of this invention specifically include, but arenot limited to, glutathione-S-transferase (GST), protein A fromStaphylococcus aureus, two synthetic IgG-binding domains (ZZ) of proteinA, outer membrane protein F, β-galactosidase (lacZ), and variousproducts of bacteriophage λ and bacteriophage T7. From the teachingsprovided herein, it is apparent that other proteins may be used ascarriers. Furthermore, the entire carrier protein need not be used, aslong as the protective anionic region is present. To facilitateisolation of the peptide sequence, amino acids susceptible to chemicalcleavage (e.g., CNBr) or enzymatic cleavage (e.g., V8 protease, trypsin)are used to bridge the peptide and fusion partner. For expression in E.coli, the fusion partner is preferably a normal intracellular proteinthat directs expression toward inclusion body formation. In such a case,following cleavage to release the final product, there is no requirementfor renaturation of the peptide. In the present invention, the DNAcassette, comprising fusion partner and peptide gene, may be insertedinto an expression vector, which can be a plasmid, virus or othervehicle known in the art. Preferably, the expression vector is a plasmidthat contains an inducible or constitutive promoter to facilitate theefficient transcription of the inserted DNA sequence in the host.Transformation of the host cell with the recombinant DNA may be carriedout by Ca⁺⁺-mediated techniques, by electroporation, or other methodswell known to those skilled in the art.

Briefly, a DNA fragment encoding a peptide is derived from an existingcDNA or genomic clone or synthesized. A convenient method isamplification of the gene from a single-stranded template. The templateis generally the product of an automated oligonucleotide synthesis.Amplification primers are derived from the 5′ and 3′ ends of thetemplate and typically incorporate restriction sites chosen with regardto the cloning site of the vector. If necessary, translationalinitiation and termination codons can be engineered into the primersequences. The sequence encoding the protein may be codon-optimized forexpression in the particular host. Thus, for example, if the analoguefusion protein is expressed in bacteria, codons are optimized forbacterial usage. Codon optimization is accomplished by automatedsynthesis of the entire gene or gene region, ligation of multipleoligonucleotides, mutagenesis of the native sequence, or othertechniques known to those in the art.

At minimum, the expression vector should contain a promoter sequence.However, other regulatory sequences may also be included. Such sequencesinclude an enhancer, ribosome binding site, transcription terminationsignal sequence, secretion signal sequence, origin of replication,selectable marker, and the like. The regulatory sequences areoperationally associated with one another to allow transcription andsubsequent translation. In preferred aspects, the plasmids used hereinfor expression include a promoter designed for expression of theproteins in bacteria. Suitable promoters, including both constitutiveand inducible promoters, are widely available and are well known in theart. Commonly used promoters for expression in bacteria includepromoters from T7, T3, T5, and SP6 phages, and the trp, lpp, and lacoperons. Hybrid promoters (see, U.S. Pat. No. 4,551,433), such as tacand trc, may also be used.

Within a preferred embodiment, the vector is capable of replication inbacterial cells. Thus, the vector may contain a bacterial origin ofreplication. Preferred bacterial origins of replication include f1-oriand col E1 ori, especially the on derived from pUC plasmids. Low copynumber vectors (e.g., pPD100) may also be used, especially when theproduct is deleterious to the host.

The plasmids also preferably include at least one selectable marker thatis functional in the host. A selectable marker gene confers a phenotypeon the host that allows transformed cells to be identified and/orselectively grown. Suitable selectable marker genes for bacterial hostsinclude the chloroamphenicol resistance gene (Cm^(r)), ampicillinresistance gene (Amp^(r)), tetracycline resistance gene (Tc^(r))kanamycin resistance gene (Kan^(r)), and others known in the art. Tofunction in selection, some markers may require a complementarydeficiency in the host.

In some aspects, the sequence of nucleotides encoding the peptide alsoencodes a secretion signal, such that the resulting peptide issynthesized as a precursor protein, which is subsequently processed andsecreted. The resulting secreted protein may be recovered from theperiplasmic space or the fermentation medium. Sequences of secretionsignals suitable for use are widely available and are well known (vonHeijne, J. Mol. Biol. 184:99-105, 1985).

The vector may also contain a gene coding for a repressor protein, whichis capable of repressing the transcription of a promoter that contains arepressor binding site. Altering the physiological conditions of thecell can depress the promoter. For example, a molecule may be added thatcompetitively binds the repressor, or the temperature of the growthmedia may be altered. Repressor proteins include, but are not limited tothe E. coli lacl repressor (responsive to induction by IPTG), thetemperature sensitive λcl857 repressor, and the like.

Examples of plasmids for expression in bacteria include the pETexpression vectors pET3a, pET 11a, pET 12a-c, and pET 15b (see U.S. Pat.No. 4,952,496; available from Novagen, Madison, Wis.). Low copy numbervectors (e.g., pPD100) can be used for efficient overproduction ofpeptides deleterious to the E. coli host (Dersch et al., FEMS Microbiol.Lett. 123: 19, 1994).

Bacterial hosts for the T7 expression vectors may contain chromosomalcopies of DNA encoding T7 RNA polymerase operably linked to an induciblepromoter (e.g., lacUV promoter; see, U.S. Pat. No. 4,952,496), such asfound in the E. coli strains HMS174(DE3)pLysS, BL21(DE3)pLysS,HMS174(DE3) and BL21(DE3). T7 RNA polymerase can also be present onplasmids compatible with the T7 expression vector. The polymerase may beunder control of a lambda promoter and repressor (e.g., pGP1-2; Taborand Richardson, Proc. Natl. Acad. Sci. USA 82: 1074, 1985).

The peptide product is isolated by standard techniques, such asaffinity, size exclusion, or ionic exchange chromatography, HPLC and thelike. An isolated peptide should preferably show a major band byCoomassie blue stain of SDS-PAGE that is at least 90% of the material.

H. Generation of Analogues by Amplification-Based Semi-RandomMutagenesis

Cationic peptide analogues can be generated using an amplification(e.g., PCR)-based procedure in which primers are designed to targetsequences at the 5′ and 3′ ends of an encoded parent peptide, forexample indolicidin. Amplification conditions are chosen to facilitatemisincorporation of nucleotides by the thermostable polymerase duringsynthesis. Thus, random mutations are introduced in the originalsequence, some of which result in amino acid alteration(s).Amplification products may be cloned into a coat protein of a phagevector, such as a phagemid vector, packaged and amplified in anacceptable host to produce a display library.

These libraries can then be assayed for antibiotic activity of thepeptides. Briefly, bacteria infected with the library are plated, grown,and overlaid with agarose containing a bacterial strain that the phageare unable to infect. Zones of growth inhibition in the agarose overlayare observed in the area of phage expressing an analogue withanti-bacterial activity. These inhibiting phage are isolated and thecloned peptide sequence determined by DNA sequence analysis. The peptidecan then be independently synthesized and its antibiotic activityfurther investigated.

5. Antibodies to Cationic Peptides

Antibodies may be generated to a specific peptide analogue usingmultiple antigenic peptides (MAPs) that contain approximately eightcopies of the peptide linked to a small non-immunogenic peptidyi core toform an immunogen. (See, in general, Harlow and Lane, supra)Alternatively, the target peptide can be conjugated to bovine serumalbumin (BSA), ovalbumin or another suitable conjugate. The MAP orpeptide conjugate is injected subcutaneously into rabbits or into miceor other rodents, where they may have sufficiently long half-lives tofacilitate antibody production. After twelve weeks blood samples aretaken, serum is separated and tested in an ELISA assay against theoriginal peptide, with a positive result indicating the presence ofantibodies specific to the target peptide. This serum can then be storedand used in ELISA assays to specifically measure the amount of thespecific analogue. Alternatively, other standard methods of antibodyproduction may be employed, for example generation of monoclonalantibodies.

Within the context of the present invention, antibodies are understoodto include monoclonal antibodies, polyclonal antibodies, anti-idiotypicantibodies, antibody fragments (e.g., Fab, and F(ab′)₂, F_(v) variableregions, or complementarity determining regions). Antibodies aregenerally accepted as specific against indolicidin analogues if theybind with a K_(d) of greater than or equal to 10⁻⁷ M, preferably greaterthan of equal to 10⁻⁸ M. The affinity of a monoclonal antibody orbinding partner can be readily determined by one of ordinary skill inthe art (see Scatchard, Ann. N.Y. Acad. Sci. 51:660-672, 1949). Oncesuitable antibodies have been obtained, they may be isolated or purifiedby many techniques well known to those of ordinary skill in the art.

Monoclonal antibodies may also be readily generated from hybridoma celllines using conventional techniques (see U.S. Pat. Nos. RE 32,011,4,902,614, 4,543,439, and 4,411,993; see also Antibodies: A LaboratoryManual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press,1988). Briefly, within one embodiment, a subject animal such as a rat ormouse is injected with peptide, generally administered as an emulsion inan adjuvant such as Freund's complete or incomplete adjuvant in order toincrease the immune response. The animal is generally boosted at leastonce prior to harvest of spleen and/or lymph nodes and immortalizationof those cells. Various immortalization techniques, such as mediated byEpstein-Barr virus or fusion to produce a hybridoma, may be used. In apreferred embodiment, immortalization occurs by fusion with a suitablemyeloma cell line to create a hybridoma that secretes monoclonalantibody. Suitable myeloma lines include, for example, NS-1 (ATCC No.TIB 18), and P3X63 Ag 8.653 (ATCC No. CRL 1580). The preferred fusionpartners do not express endogenous antibody genes. After about sevendays, the hybridomas may be screened for the presence of antibodies thatare reactive against a telomerase protein. A wide variety of assays maybe utilized (see Antibodies: A Laboratory Manual, Harlow and Lane(eds.), Cold Spring Harbor Laboratory Press, 1988).

Other techniques may also be utilized to construct monoclonal antibodies(see Huse et al., Science 246:1275-1281, 1989; Sastry et al., Proc.Natl. Acad. Sci. USA 86:5728-5732, 1989; Alting-Mees et al., Strategiesin Molecular Biology 3:1-9, 1990; describing recombinant techniques).These techniques include cloning heavy and light chain immunoglobulincDNA in suitable vectors, such as λImmunoZap(H) and λImmunoZap(L). Theserecombinants may be screened individually or co-expressed to form Fabfragments or antibodies (see Huse et al., supra; Sastry et al., supra).Positive plaques may subsequently be converted to a non-lytic plasmidthat allows high level expression of monoclonal antibody fragments fromE. coli.

Similarly, portions or fragments, such as Fab and Fv fragments, ofantibodies may also be constructed utilizing conventional enzymaticdigestion or recombinant DNA techniques to yield isolated variableregions of an antibody. Within one embodiment, the genes which encodethe variable region from a hybridoma producing a monoclonal antibody ofinterest are amplified using nucleotide primers for the variable region.In addition, techniques may be utilized to change a “murine” antibody toa “human” antibody, without altering the binding specificity of theantibody.

II. Testing

Cationic peptides of the present invention are assessed either alone orin combination with an antibiotic agent or another analogue for theirpotential as antibiotic therapeutic agents using a series of assays.Preferably, all peptides are initially assessed in vitro, the mostpromising candidates are selected for further assessment in vivo, andthen candidates are selected for pre-clinical studies. The in vitroassays include measurement of antibiotic activity, toxicity, solubility,pharmacology, secondary structure, liposome permeabilization and thelike. In vivo assays include assessment of efficacy in animal models,antigenicity, toxicity, and the like. In general, in vitro assays areinitially performed, followed by in vivo assays.

Generally, cationic peptides are initially tested for (1) anti-microbialactivity in vitro; (2) in vitro toxicity to normal mammalian cells; and(3) in vivo toxicity in an animal model. Peptides that have someanti-microbial activity are preferred, although such activity may not benecessary for enhancing the activity of an antibiotic agent. Also, forin vivo use, peptides should preferably demonstrate acceptable toxicityprofiles, as measured by standard procedures. Lower toxicity ispreferred. Additional assays may be performed to demonstrate that thepeptide is not immunogenic and to examine antimicrobial activity invivo.

A. In Vitro Assays

Cationic peptides, including indolicidin analogues, are assayed by, forexample, an agarose dilution MIC assay, a broth dilution, time-killassay, or equivalent methods. Antibiotic activity is measured asinhibition of growth or killing of a microorganism (e.g., bacteria,fungi).

Briefly, a candidate peptide in Mueller Hinton broth supplemented withcalcium and magnesium is mixed with molten agarose. Other broths andagars may be used as long as the peptide can freely diffuse through themedium. The agarose is poured into petri dishes or wells, allowed tosolidify, and a test strain is applied to the agarose plate. The teststrain is chosen, in part, on the intended application of the peptide.Thus, by way of example, if an indolicidin analogue with activityagainst S. aureus is desired, an S. aureus strain is used. It may bedesirable to assay the analogue on several strains and/or on clinicalisolates of the test species. Plates are incubated overnight andinspected visually for bacterial growth. A minimum inhibitoryconcentration (MIC) of a cationic peptide is the lowest concentration ofpeptide that completely inhibits growth of the organism. Peptides thatexhibit good activity against the test strain, or group of strains,typically having an MIC of less than or equal to 16 □g/ml are selectedfor further testing.

Alternatively, time kill curves can be used to determine the differencesin colony counts over a set time period, typically 24 hours. Briefly, asuspension of organisms of known concentration is prepared and acandidate peptide is added. Aliquots of the suspension are removed atset times, diluted, plated on medium, incubated, and counted. MIC ismeasured as the lowest concentration of peptide that completely inhibitsgrowth of the organism. In general, lower MIC values are preferred.

Candidate cationic peptides may be further tested for their toxicity tonormal mammalian cells. An exemplary assay is a red blood cell (RBC)(erythrocyte) hemolysis assay. Briefly, in this assay, red blood cellsare isolated from whole blood, typically by centrifugation, and washedfree of plasma components. A 5% (v/v) suspension of erythrocytes inisotonic saline is incubated with different concentrations of peptideanalogue. Generally, the peptide will be in a suitable formulationbuffer. After incubation for approximately 1 hour at 37° C., the cellsare centrifuged, and the absorbance of the supernatant at 540 nm isdetermined. A relative measure of lysis is determined by comparison toabsorbance after complete lysis of erythrocytes using NH₄Cl orequivalent (establishing a 100% value). A peptide with <10% lysis at 100μg/ml is suitable. Preferably, there is <5% lysis at 100 μg/ml. Suchpeptides that are not lytic, or are only moderately lytic, are desirableand suitable for further screening. Other in vitro toxicity assays, forexample measurement of toxicity towards cultured mammalian cells, may beused to assess in vitro toxicity.

Solubility of the peptide in formulation buffer is an additionalparameter that may be examined. Several different assays may be used,such as appearance in buffer. Briefly, peptide is suspended in solution,such as broth or formulation buffer. The appearance is evaluatedaccording to a scale that ranges from (a) clear, no precipitate, (b)light, diffuse precipitate, to (c) cloudy, heavy precipitate. Finergradations may be used. In general, less precipitate is more desirable.However, some precipitate may be acceptable.

Additional in vitro assays may be carried out to assess the potential ofthe peptide as a therapeutic. Such assays include peptide solubility informulations, pharmacology in blood or plasma, serum protein binding,analysis of secondary structure, for example by circular dichroism,liposome permeabilization, and bacterial inner membranepermeabilization. In general, it is desirable that analogues are solubleand perform better than the parent peptide (e.g., indolicidin).

B. In Vivo Assays

Peptides, including peptide analogues, selected on the basis of theresults from the in vitro assays can be tested in vivo for efficacy,toxicity and the like.

The antibiotic activity of selected peptides may be assessed in vivo fortheir ability to ameliorate microbial infections using animal models. Avariety of methods and animal models are available. Within these assays,a peptide is useful as a therapeutic if inhibition of microorganismalgrowth compared to inhibition with vehicle alone is statisticallysignificant. This measurement can be made directly from culturesisolated from body fluids or sites, or indirectly, by assessing survivalrates of infected animals. For assessment of antibacterial activityseveral animal models are available, such as acute infection modelsincluding those in which (a) normal mice receive a lethal dose ofmicroorganisms, (b) neutropenic mice receive a lethal dose ofmicroorganisms or (c) rabbits receive an inoculum in the heart, andchronic infection models. The model selected will depend in part on theintended clinical indication of the analogue.

By way of example, in a normal mouse model, mice are inoculated ip or ivwith a lethal dose of bacteria. Typically, the dose is such that 90-100%of animals die within 2 days. The choice of a microorganismal strain forthis assay depends, in part, upon the intended application of theanalogue, and in the accompanying examples, assays are carried out withthree different Staphylococcus strains. Briefly, shortly before or afterinoculation (generally within 60 minutes), analogue in a suitableformulation buffer is injected. Multiple injections of analogue may beadministered. Animals are observed for up to 8 days post-infection andthe survival of animals is recorded. Successful treatment either rescuesanimals from death or delays death to a statistically significant level,as compared with non-treatment control animals. Analogues that showbetter efficacy than indolicidin itself are preferred.

In vivo toxicity of a peptide is measured through administration of arange of doses to animals, typically mice, by a route defined in part bythe intended clinical use. The survival of the animals is recorded andLD₅₀, LD₉₀₋₁₀₀, and maximum tolerated dose (MTD) can be calculated toenable comparison of analogues. Indolicidin analogues less toxic thanindolicidin are preferred.

Furthermore, for in vivo use, low immunogenicity is preferred. Tomeasure immunogenicity, peptides are injected into normal animals,generally rabbits. At various times after a single or multipleinjections, serum is obtained and tested for antibody reactivity to thepeptide analogue. Antibodies to peptides may be identified by ELISA,immunoprecipitation assays, Western blots, and other methods. (see,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1988). No or minimalantibody reactivity is preferred. Additionally, pharmacokinetics of theanalogues in animals and histopathology of animals treated withanalogues may be determined.

Selection of cationic peptides as potential therapeutics is based on invitro and in vivo assay results. In general, peptides that exhibit lowtoxicity at high dose levels and high efficacy at low dose levels arepreferred candidates.

III. Antibiotic Agents

An antibiotic agent includes any molecule that tends to prevent, inhibitor destroy life and as such, includes anti-bacterial agents,anti-fungicides, anti-viral agents, and anti-parasitic agents. Theseagents may be isolated from an organism that produces the agent orprocured from a commercial source (e.g., pharmaceutical company, such asEli Lilly, Indianapolis, Ind.; Sigma, St. Louis, Mo.).

Anti-bacterial antibiotic agents include, but are not limited to,penicillins, cephalosporins, carbacephems, cephamycins, carbapenems,monobactams, aminoglycosides, glycopeptides, quinolones, tetracyclines,macrolides, and fluoroquinolones (see Table below). Examples ofantibiotic agents include, but are not limited to, Penicillin G (CASRegistry No.: 61-33-6); Methicillin (CAS Registry No.: 61-32-5);Nafcillin (CAS Registry No.: 147-52-4); Oxacillin (CAS Registry No.:66-79-5); Cloxacillin (CAS Registry No.: 61-72-3); Dicloxacillin (CASRegistry No.: 3116-76-5); Ampicillin (CAS Registry No.: 69-53-4);Amoxicillin (CAS Registry No.: 26787-78-0); Ticarcillin (CAS RegistryNo.: 34787-01-4); Carbenicillin (CAS Registry No.: 4697-36-3);Mezlocillin (CAS Registry No.: 51481-65-3); Azlocillin (CAS RegistryNo.: 37091-66-0); Piperacillin (CAS Registry No.: 61477-96-1); Imipenem(CAS Registry No.: 74431-23-5); Aztreonam (CAS Registry No.:78110-38-0); Cephalothin (CAS Registry No.: 153-61-7); Cefazolin (CASRegistry No.: 25953-19-9); Cefaclor (CAS Registry No.: 70356-03-5);Cefamandole formate sodium (CAS Registry No.: 42540-40-9); Cefoxitin(CAS Registry No.: 35607-66-0); Cefuroxime (CAS Registry No.:55268-75-2); Cefonicid (CAS Registry No.: 61270-58-4); Cefmetazole (CASRegistry No.: 56796-20-4); Cefotetan (CAS Registry No.: 69712-56-7);Cefprozil (CAS Registry No.: 92665-29-7); Loracarbef (CAS Registry No.:121961-22-6); Cefetamet (CAS Registry No.: 65052-63-3); Cefoperazone(CAS Registry No.: 62893-19-0); Cefotaxime (CAS Registry No.:63527-52-6); Ceftizoxime (CAS Registry No.: 68401-81-0); Ceftriaxone(CAS Registry No.: 73384-59-5); Ceftazidime (CAS Registry No.:72558-82-8); Cefepime (CAS Registry No.: 88040-23-7); Cefixime (CASRegistry No.: 79350-37-1); Cefpodoxime (CAS Registry No.: 80210-62-4);Cefsulodin (CAS Registry No.: 62587-73-9); Fleroxacin (CAS Registry No.:79660-72-3); Nalidixic acid (CAS Registry No.: 389-08-2); Norfloxacin(CAS Registry No.: 70458-96-7); Ciprofloxacin (CAS Registry No.:85721-33-1); Ofloxacin (CAS Registry No.: 82419-36-1); Enoxacin (CASRegistry No.: 74011-58-8); Lomefloxacin (CAS Registry No.: 98079-51-7);Cinoxacin (CAS Registry No.: 28657-80-9); Doxycycline (CAS Registry No.:564-25-0); Minocycline (CAS Registry No.: 10118-90-8); Tetracycline (CASRegistry No.: 60-54-8); Amikacin (CAS Registry No.: 37517-28-5);Gentamicin (CAS Registry No.: 1403-66-3); Kanamycin (CAS Registry No.:8063-07-8); Netilmicin (CAS Registry No.: 56391-56-1); Tobramycin (CASRegistry No.: 32986-56-4); Streptomycin (CAS Registry No.: 57-92-1);Azithromycin (CAS Registry No.: 83905-01-5); Clarithromycin (CASRegistry No.: 81103-11-9); Erythromycin (CAS Registry No.: 114-07-8);Erythromycin estolate (CAS Registry No.: 3521-62-8); Erythromycin ethylsuccinate (CAS Registry No.: 41342-53-4); Erythromycin glucoheptonate(CAS Registry No.: 23067-13-2); Erythromycin lactobionate (CAS RegistryNo.: 3847-29-8); Erythromycin stearate (CAS Registry No.: 643-22-1);Vancomycin (CAS Registry No.: 1404-90-6); Teicoplanin (CAS Registry No.:61036-64-4); Chloramphenicol (CAS Registry No.: 56-75-7); Clindamycin(CAS Registry No.: 18323-44-9); Trimethoprim (CAS Registry No.:738-70-5); Sulfamethoxazole (CAS Registry No.: 723-46-6); Nitrofurantoin(CAS Registry No.: 67-20-9); Rifampin (CAS Registry No.: 13292-46-1);Mupirocin (CAS Registry No.: 12650-69-0); Metronidazole (CAS RegistryNo.: 443-48-1); Cephalexin (CAS Registry No.: 15686-71-2); Roxithromycin(CAS Registry No.: 80214-83-1); Co-amoxiclavuanate; combinations ofPiperacillin and Tazobactam; and their various salts, acids, bases, andother derivatives.

A table presenting categories of antibiotics, their mode of action, andexamples of antibiotics is shown below.

TABLE 2 Class of Antibiotic Antibiotic Mode of Action PENICILLINS Blocksthe formation of Natural Penicillin G, Benzylpenicillin new cell wallsin bacteria Penicillin V, Phenoxymethylpenicillin Penicillinaseresistant Methicillin, Nafcillin, Oxacillin Cloxacillin, DicloxacillinAcylamino-penicillins Ampicillin, Amoxicillin Carboxy-penicillinsTicarcillin, Carbenicillin Ureido-penicillins Mezlocillin, Azlocillin,Piperacillin CARBAPENEMS Imipenem, Meropenem Blocks the formation of newcell walls in bacteria MONOBACTAMS Aztreonam Blocks the formation of newcell walls in bacteria CEPHALOSPORINS Prevents formation of new 1stGeneration Cephalothin, Cefazolin cell walls in bacteria 2nd GenerationCefaclor, Cefamandole Cefuroxime, Cefonicid, Cefmetazole, Cefotetan,Cefprozil 3rd Generation Cefetamet, Cefoperazone Cefotaxime, CeftizoximeCeftriaxone, Ceftazidime Cefixime, Cefpodoxime, Cefsulodin 4thGeneration Cefepime CARBACEPHEMS Loracarbef Prevents formation of newcell walls in bacteria CEPHAMYCINS Cefoxitin Prevents formation of newcell walls in bacteria QUINOLONES Fleroxacin, Nalidixic Acid Inhibitsbacterial DNA Norfloxacin, Ciprofloxacin synthesis Ofloxacin, EnoxacinLomefloxacin, Cinoxacin TETRACYCLINES Doxycycline, Minocycline, Inhibitsbacterial protein Tetracycline synthesis, binds to 30S ribosome subunit.AMINOGLYCOSIDES Amikacin, Gentamicin, Kanamycin, Inhibits bacterialprotein Netilmicin, Tobramycin, synthesis, binds to 30S Streptomycinribosome subunit. MACROLIDES Azithromycin, Clarithromycin, Inhibitsbacterial protein Erythromycin synthesis, binds to 50S ribosome subunitDerivatives of Erythromycin estolate, Erythromycin Erythromycin stearateErythromycin ethylsuccinate Erythromycin gluceptate Erythromycinlactobionate GLYCOPEPTIDES Vancomycin, Teicoplanin Inhibits cell wallsynthesis, prevents peptidoglycan elongation. MISCELLANEOUSChloramphenicol Inhibits bacterial protein synthesis, binds to 50Sribosome subunit. Clindamycin Inhibits bacterial protein synthesis,binds to 50S ribosome subunit. Trimethoprim Inhibits the enzymedihydrofolate reductase, which activates folic acid. SulfamethoxazoleActs as antimetabolite of PABA & inhibits synthesis of folic acidNitrofurantoin Action unknown, but is concentrated in urine where it canact on urinary tract bacteria Rifampin Inhibits bacterial RNA polymeraseMupirocin Inhibits bacterial protein synthesis

Anti-fungal agents include, but are not limited to, terbinafinehydrochloride, nystatin, amphotericin B, griseofulvin, ketoconazole,miconazole nitrate, flucytosine, fluconazole, itraconazole,clotrimazole, benzoic acid, salicylic acid, and selenium sulfide.

Anti-viral agents include, but are not limited to, amantadinehydrochloride, rimantadin, acyclovir, famciclovir, foscarnet,ganciclovir sodium, idoxuridine, ribavirin, sorivudine, trifluridine,valacyclovir, vidarabin, didanosine, stavudine, zalcitabine, zidovudine,interferon alpha, and edoxudine.

Anti-parasitic agents include, but are not limited to,pirethrins/piperonyl butoxide, permethrin, iodoquinol, metronidazole,diethylcarbamazine citrate, piperazine, pyrantel pamoate, mebendazole,thiabendazole, praziquantel, albendazole, proguanil, quinidine gluconateinjection, quinine sulfate, chloroquine phosphate, mefloquinehydrochloride, primaquine phosphate, atovaquone, co-trimoxazole(sulfamethoxazole/trimethoprim), and pentamidine isethionate.

IV. Enhanced Activity of Combinations of Cationic Peptides andAntibiotic Agents

Enhanced activity occurs when a combination of peptide and antibioticagent potentiates activity beyond the individual effects of the peptideor antibiotic agent alone or additive effects of peptide plus antibioticagent. Enhanced activity is especially desirable in at least fourscenarios: (1) the microorganism is sensitive to the antibiotic agent,but the dosage has associated problems; (2) the microorganism istolerant to the antibiotic agent, and is inhibited from growing but isnot killed; (3) the microorganism is inherently resistant to theantibiotic agent; and (4) the microorganism has acquired resistance tothe antibiotic agent. Enhanced efficacy resulting from administration ofthe antibiotic agent in combination with a cationic peptide in the abovescenarios: (1) allows for administration of lower dosages or antibioticagent and cationic peptide; (2) restores a cytocidal effect; (3)overcomes inherent resistance; and (4) overcomes acquired resistance.

A. Enhancement of Antibiotic Agent or Cationic Peptide Activity

A synergistic combination of cationic peptide and antibiotic agent maypermit a reduction in the dosage of one or both agents in order toachieve a similar therapeutic effect. This would allow smaller doses tobe used, thus, decreasing the incidence of toxicity (e.g., fromaminoglycosides) and lowering costs of expensive antibiotics (e.g.,vancomycin). Concurrent or sequential administration of peptide andantibiotic agent is expected to provide more effective treatment ofinfections caused by micro-organisms (bacteria, viruses, fungi, andparasites). In particular, this could be achieved by using doses thatthe peptide or antibiotic agent alone would not achieve therapeuticsuccess. Alternatively, the antibiotic agent and peptide can beadministered at therapeutic doses for each, but wherein the combinationof the two agents provides even more potent effects.

As used herein, “synergy” refers to the in vitro effect ofadministration of a combination of a cationic peptide and antibioticagent such that (1) the fractional inhibitory concentration (FIC) isless than or equal to 0.5 in an FIC assay described herein; or (2) thereis at least a 100-fold (2 log₁₀) increase in killing at 24 hours for thecombination as compared with the antibiotic agent alone in a time killcurve assay as described herein.

Such synergy is conveniently measured in an in vitro assay, such askinetic kill studies or a fractional inhibitory concentration (FIC)assay as determined by agarose or broth dilution assay. The agarosedilution assay is preferred.

Briefly, in the dilution assay, a checkerboard array of cationicpeptides and antibiotic agents titrated in doubling dilutions areinoculated with a microbial (e.g., bacterial) isolate. The FIC isdetermined by observing the impact of one antibiotic agent on the MIC(“minimal inhibitory concentration”) of the cationic peptide and viceversa. FIC is calculated by the following formula:

${FIC} = {\frac{{MIC}\left( {{peptide}\mspace{14mu} {in}\mspace{14mu} {combination}} \right)}{{MIC}\left( {{peptide}\mspace{14mu} {alone}} \right)} + \frac{{MIC}\left( {{antibiotic}\mspace{14mu} {in}\mspace{14mu} {combination}} \right)}{{MIC}\left( {{antibiotic}\mspace{14mu} {alone}} \right)}}$

An FIC of ≦0.5 is evidence of synergy. An additive response has an FICvalue of >0.5 and less than or equal to 1, while an indifferent responsehas an FIC value of >1 and ≦2. Although a synergistic effect ispreferred, an additive effect may still indicate that the combination ofantibiotic agent and cationic peptide are therapeutically useful.

B. Overcoming Tolerance

Tolerance is associated with a defect in bacterial cellular autolyticenzymes such that an antibacterial agent demonstrates bacteriostaticrather than bactericidal activity (Mahon and Manuselis, Textbook ofDiagnostic Microbiology, W.B. Saunders Co., Toronto, Canada, p. 92,1995). For antibiotic agents that have only bacteriostatic activity, theadministration of cationic peptides in combination with antibioticagents can restore bactericidal activity. Alternatively, the addition ofa peptide to an antibiotic agent may increase the rate of a bactericidaleffect of an antibiotic.

Bactericidal effects of antibiotics can be measured in vitro by avariety of assays. Typically, the assay is a measurement of MBC(“minimal bactericidal concentration”), which is an extension of the MICdetermination. The agarose dilution assay is adapted to provide both MBCand MIC for an antimicrobial agent alone and the agent in combinationwith a cationic peptide. Alternatively, kinetic time-kill (or growth)curves can be used to determine MIC and MBC.

Briefly, following determination of MIC, MBC is determined from theassay plates by swabbing the inocula on plates containing antibioticagent in concentrations at and above the MIC, resuspending the swab insaline or medium, and plating an aliquot on agarose plates. If thenumber of colonies on these agarose plates is less than 0.1% of theinitial inoculum (as determined by a plate count immediately afterinoculation of the MIC test plates), then ≧99.9% killing has occurred.The MBC end point is defined as the lowest concentration of theantimicrobial agent that kills 99.9% of the test bacteria.

Thus, tolerance of a microorganism to an antimicrobial agent isindicated when the number of colonies growing on subculture platesexceeds the 0.1% cutoff for several successive concentrations above theobserved MIC. A combination of antimicrobial agent and cationic peptidethat breaks tolerance results in a decrease in the MBC:MIC ratio to <32.

C. Overcoming Inherent Resistance

The combination of a cationic peptide with an antibiotic agent, forwhich a microorganism is inherently resistant (i.e., the antibiotic hasnever been shown to be therapeutically effective against the organism inquestion), is used to overcome the resistance and confer susceptibilityof the microorganism to the agent. Overcoming inherent resistance isespecially useful for infections where the causative organism isbecoming or has become resistant to most, if not all, of the currentlyprescribed antibiotics. Additionally, administering a combinationtherapy provides more options when toxicity of an antibiotic agentand/or price are a consideration.

Overcoming resistance can be conveniently measured in vitro. Resistanceis overcome when the MIC for a particular antibiotic agent against aparticular microorganism is decreased from the resistant range to thesensitive range (according to the National Committee for ClinicalLaboratory Standards (NCCLS)) (see also, Moellering, in Principles andPractice of Infectious Diseases, 4th edition, Mandell et al., eds.Churchill Livingstone, NY, 1995). NCCLS standards are based onmicrobiological data in relation to pharmacokinetic data and clinicalstudies. Resistance is determined when the organism causing theinfection is not inhibited by the normal achievable serum concentrationsof the antibiotic agent based on recommended dosage. Susceptibility isdetermined when the organism responds to therapy with the antibioticagent used at the recommended dosage for the type of infection andmicroorganism.

D. Overcoming Acquired Resistance

Acquired resistance in a microorganism that was previously sensitive toan antibiotic agent is generally due to mutational events in chromosomalDNA, acquisition of a resistance factor carried via plasmids or phage,or transposition of a resistance gene or genes from a plasmid or phageto chromosomal DNA.

When a microorganism acquires resistance to an antibiotic, thecombination of a peptide and antibiotic agent can restore activity ofthe antibiotic agent by overcoming the resistance mechanism of theorganism. This is particularly useful for organisms that are difficultto treat or where current therapy is costly or toxic. The ability to usea less expensive or less toxic antibiotic agent, which had beeneffective in the past, is an improvement for certain current therapies.The re-introduction of an antibiotic agent would enable previousclinical studies and prescription data to be used in its evaluation.Activity is measured in vitro by MICs or kinetic kill curves and in vivousing animal and human clinical trials.

E. Enhancement of Effect of Lysozyme and Nisin

The combination of cationic peptides and lysozyme or nisin may improvetheir antibacterial effectiveness and allow use in situations in whichthe single agent is inactive or inappropriate.

Lysozymes disrupt certain bacteria by cleaving the glycosidic bondbetween N-acetylglucosamine and N-acetylmuramic acid in thepolysaccharide component of bacterial cell walls. However, lysozymeexhibits only weak antibacterial activity with a narrow spectrum ofactivity. The addition of cationic peptide may improve the effectivenessof this activity and broaden the spectrum of activity.

Nisins are 34-residue peptide lantibiotics with primarilyanti-Gram-positive bacterial activity. Nisin is used in the foodprocessing industry as a preservative, especially for cheese, cannedfruits and vegetables. Nisin forms transient potential-dependent poresin the bacterial cytoplasmic membranes but also exhibits weakantibacterial activity with a narrow spectrum of activity. The additionof cationic peptide may improve the effectiveness of nisin and broadenthe spectrum of activity.

F. In Vivo Assays

In vivo testing involves the use of animal models of infection.Typically, but not exclusively, mice are used. The test organism ischosen according to the intended combination of cationic peptide andantibiotic to be evaluated. Generally, the test organism is injectedintraperitoneally (IP) or intravenously (IV) at 10 to 100 times thefifty percent lethal dose (LD₅₀). The LD₅₀ is calculated using a methoddescribed by Reed and Muench (Reed L J and Muench H. The AmericanJournal of Hygiene, 27:493-7.). The antibiotic agent and the cationicpeptide are injected IP, IV, or subcutaneously (SC) individually as wellas in combination to different groups of mice. The antimicrobial agentsmay be given in one or multiple doses. Animals are observed for 5 to 7days. Other models of infection may also be used according to theclinical indication for the combination of antibiotic agents.

The number of mice in each group that survive the infectious insult isdetermined after 5 to 7 days. In addition, when bacteria are the testorganisms, bacterial colony counts from blood, peritoneal lavage fluid,fluid from other body sites, and/or tissue from different body sitestaken at various time intervals can be used to assess efficacy. Samplesare serially diluted in isotonic saline and incubated for 20-24 hours,at 37° C., on a suitable growth medium for the bacterium.

Synergy between the cationic peptide and the antibiotic agent isassessed using a model of infection as described above. For adetermination of synergy, one or more of the following should occur. Thecombination group should show greater survival rates compared to thegroups treated with only one agent; the combination group and theantibiotic agent group have equivalent survival rates with thecombination group receiving a lower concentration of antibiotic agent;the combination group has equivalent or better survival compared to anantibiotic agent group with a lower microorganismal load at various timepoints.

Overcoming tolerance can be demonstrated by lower bacterial colonycounts at various time points in the combination group over theantibiotic agent group. This may also result in better survival ratesfor the combination group.

Similar animal models to those described above can be used to establishwhen inherent or acquired resistance is overcome. The microorganismstrain used is, by definition, resistant to the antibiotic agent and sothe survival rate in the antibiotic agent group will be close, if notequal, to zero percent. Thus, overcoming the inherent resistance of themicroorganism to the antibiotic agent is demonstrated by increasedsurvival of the combination group. Testing for reversing acquiredresistance may be performed in a similar manner.

V. Combinations of Peptides and Antibiotic Agents

As discussed herein, cationic peptides are administered in combinationwith antibiotic agents. The combination enhances the activity of theantibiotic agents. Such combinations may be used to effect a synergisticresult, overcome tolerance, overcome inherent resistance, or overcomeacquired resistance of the microorganism to the antibiotic agent.

To achieve a synergistic effect, a combination of antibiotic agent andcationic peptide is administered to a patient or administered in such amanner as to contact the microorganism. Any combination of antibioticagent and cationic peptide may result in a synergistic effect and, thus,is useful within the context of this invention.

In particular, certain microorganisms are preferred targets. Inconjunction with these microorganisms, certain commonly used antibioticagents are preferred to be enhanced. The table below sets out thesemicroorganisms, antibiotic agents, and cationic peptide combinationsthat are preferred.

TABLE 3 BACTERIAL SPECIES ANTIMICROBIAL AGENTS PEPTIDE A. baumanniiGentamicin MBI 21A2 B. cepacia Ceftriaxone MBI 11J02CN E. cloacaeCiprofloxacin MBI 29A2 E. faecalis Amikacin MBI 11B16CN E. faeciumVancomycin MBI 29 P. aeruginosa Mupirocin MBI 28 P. aeruginosaTobramycin MBI 11G13CN S marcescens Piperacillin MBI 11G7CN S. aureusPiperacillin MBI 11CN S. maltophilia Tobramycin REWH 53A5CN MYCOSESANTIFUNGAL AGENTS PEPTIDE Candida species Fluconazole MBI 28Cryptococcus Fluconazole MBI 29A3 Aspergillus species Itraconazole MBI26 VIRUSES ANTIVIRAL AGENTS PEPTIDE Herpes simplex virus Acyclovir MBI11A2CN Influenza A virus Amantadine- MBI 21A1 rimantadine PARASITESANTIPARASITIC AGENTS PEPTIDE Trichomonas vaginalis Metronidazole MBI 29Plasmodium Chloroquine MBI 11D18CN falciparum

To overcome tolerance, a combination of antibiotic agent and cationicpeptide is administered to a patient or administered in such a manner asto contact the microorganism. Any combination of antibiotic agent andcationic peptide that overcomes tolerance is useful within the contextof this invention. In particular, certain microorganisms, which exhibittolerance to specific antibiotic agents are preferred targets. The tablebelow sets out these microorganisms, antibiotic agents, and cationicpeptide combinations that are preferred.

TABLE 4 BACTERIAL SPECIES ANTIMICROBIAL AGENTS PEPTIDE Enterococcusspecies Ampicillin (Amino-penicillins) MBI 21A10 Piperacillin(Penicillins, antipseudomonal) Enterococcus species Gentamicin(Aminoglycosides) MBI 29 Enterococcus species Vancomycin, TeicoplaninMBI 26 (glycopeptides) Streptococcus Penicillins MBI 29A3 pneumoniaeSalmonella typhi Chloramphenicol MBI 11A1CN Campylobacter jejuniErythromycin (Macrolides) MBI 11B4CN

To overcome inherent resistance, a combination of antibiotic agent andcationic peptide is administered to a patient or administered in such amanner as to contact the microorganism. Any combination of antibioticagent and cationic peptide that overcomes resistance is useful withinthe context of this invention. In particular, certain microorganisms,which exhibit inherent resistance to specific antibiotic agents arepreferred targets. The table below sets out these microorganisms,antibiotic agents, and cationic peptide combinations that are preferred.

TABLE 5 BACTERIAL SPECIES ANTIMICROBIAL AGENTS PEPTIDEMethicillin-resistant Amikacin MBI 29F1 S. aureus S. maltophiliaGentamicin MBI 11D18CN S. maltophilia Gentamicin MBI 26 S. maltophiliaTobramycin MBI 29A3 Methicillin-resistant Tobramycin MBI 21A1 S. aureusE. coli Mupirocin MBI 21A1 S. maltophilia Amikacin MBI 11B16CN S.maltophilia Amikacin MBI 26 B. cepacia Amikacin MBI 29A3 Methicillinresistant Gentamicin MBI 11D18CN S. aureus MYCOSES ANTIFUNGAL AGENTSPEPTIDE Aspergillosis Fluconazole MBI 11D18CN Candida speciesGriseofulvin MBI 29

To overcome acquired resistance, a combination of antibiotic agent andcationic peptide is administered to a patient or administered in such amanner as to contact the microorganism. Any combination of antibioticagent and cationic peptide that overcomes resistance is useful withinthe context of this invention. In particular, certain microorganisms,which exhibit acquired resistance to specific antibiotic agents arepreferred targets. The table below sets out these microorganisms,antibiotic agents, and cationic peptide combinations that are preferred.

TABLE 6 BACTERIA ANTIMICROBIAL AGENT PEPTIDE Enterococcus spp.Vancomycin MBI 26 P. aeruginosa Ceftriaxone MBI 26 S. aureusCiprofloxacin MBI 29A2 E. cloacae Piperacillin MBI 11F4CN P. aeruginosaTobramycin MBI 21A1 P. aeruginosa Ciprofloxacin MBI 29A2 P. aeruginosaGentamicin MBI 11B16CN S. epidermidis Gentamicin MBI 11D18CNAcinetobacter spp. Tobramycin MBI 11F3CN Enterococcus spp. VancomycinMBI 11A1CN MYCOSES ANTIFUNGAL AGENTS PEPTIDE Candida species FluconazoleMBI 11CN Cryptococcus Fluconazole MBI 11A1CN VIRUSES ANTIVIRAL AGENTSPEPTIDE Herpes simplex virus Acyclovir MBI 29 Respiratory SyncytialRibavirin MBI 26 Virus (RSV) Influenza A virus Amantadine-rimantadineMBI 26 PARASITES ANTIPARASITIC AGENTS PEPTIDE Trichomonas vaginalisMetronidazole MBI 29 Pneumocystis carinii Cotrimoxazole MBI 29A3Plasmodium Chloroquine MBI 26 falciparum

Additional preferred combinations for indolicidin analogues are listedbelow:

ANTIBIOTIC PEPTIDE Ciprofloxacin MBI 11A1CN Vancomycin MBI 11A1CNPiperacillin MBI 11B9CN Gentamicin MBI 11B16CN Piperacillin MBI 11D18CNTobramycin MBI 11D18CN Vancomycin MBI 11D18CN Piperacillin MBI 11E3CNTobramycin MBI 11F3CN Piperacillin MBI 11F4CN

VI. Polymer Modification of Peptides and Proteins

As noted herein, the present invention provides methods and compositionsfor modifying a compound with a free amine group. The amine group may bepart of the native structure of the compound or added by a chemicalmethod. Thus, peptides, proteins, and antibiotics and the like can bemodified with an activated polyoxyalkylene and derivatives. When thecompounds are peptides or proteins, the modified or derivatized formsare referred to herein as “APO-modified peptides” or “APO-modifiedproteins”. Similarly, modified forms of antibiotics are referred to as“APO-modified antibiotics.” APO-modified compounds (e.g., APO-cationicpeptides) generally exhibit improved pharmacological properties.

A. Characteristics of an Activated Polyoxyalkylene Reagent

As discussed herein, a suitable reagent for formation of APO-modifiedcompounds (e.g., peptides and proteins) comprises a hydrophobic regionand a hydrophilic region, and optionally a linker. The hydrophobicregion is a lipophilic compound with a suitable functional group forconjugation to the hydrophilic region or linker. The hydrophilic regionis a polyoxyalkylene. As used herein, “polyoxyalkylene” refers to 2 or 3carbon polyoxyalkylene polymers. The polymer chain is of a length 2units or greater. Two carbon polyoxyalkylenes include polyoxyethyleneand its derivatives, polyethylene glycol (PEG) of various molecularweights, and its derivatives, such as polysorbate. Three carbonpolyoxyalkylenes include polyoxypropylene and derivatives andpolypropylene glycol and its derivatives. Derivatives include alkyl- andaryl-polyoxyethylene compounds.

The hydrophobic region is a lipophilic moiety, generally a fatty acid,but may be a fatty alcohol, fatty thiol, hydrocarbons (such as4-(1,1,3,3-tetramethylbutyl)-cyclohexyl), aryl compounds (such as4-(1,1,3,3-tetramethylbutyl)-phenyl) and the like, which are alsolipophilic compounds. The fatty acid may be saturated or unsaturated.The chain length does not appear to be important, although typicallycommercially available fatty acids are used and have chain lengths ofC₁₂₋₁₈. The length may be limited however by solubility or solidity ofthe compound, that is longer lengths of fatty acids are solid at roomtemperature. Fatty acids of 12 carbons (lauryl), 14 carbons, 16 carbons(palmitate), and 18 carbons (monostearate or oleate) are preferred chainlengths.

The hydrophilic region is a polyoxyalkylene, such as polyethylene,polypropylene glycol monoether (for example Triton X114), andpolysorbate. For polysorbate, the ether function is formed by thelinkage between the polyoxyethylene chain, preferably having a chainlength of from 2 to 100 monomeric units, and the sorbitan group.Polymethylene glycol is unsuitable for administration in animals due toformation of formaldehydes, and glycols with a chain length of 4 may beinsoluble. Mixed polyoxyethylene-polyoxypropylene chains are alsosuitable.

A linker for bridging the hydrophilic and hydrophobic regions is notrequired, but if used, should be able to bridge both a polyoxyalkyleneand the hydrophobic region. Suitable linkers include sorbitan, sugaralcohols, ethanolamine, ethanolthiol, 2-mercaptoethanol,1,6-diaminohexane, an amino acid (e.g., glutamine, lysine), otherreduced sugars, and the like. For example, sorbitan forms an esterlinkage with the fatty acid in a polysorbate.

Suitable compounds include polyoxyethylenesorbitans, such as themonolaurate, monooleate, monopalmitate, monostearate, trioleate, andtristearate esters. These and other suitable compounds may besynthesized by standard chemical methods or obtained commercially (e.g.,Sigma Chemical Co., MO; Aldrich Chemical Co., Wisconsin; J. B. Baker,New Jersey).

B. Activation of Reagent

The reagent is activated by exposure to UV light with free exchange ofair or by chemical treatment with ammonium persulfate, or a combinationof these methods.

Photoactivation is achieved using a lamp that irradiates at 254 nm or302 nm. Preferably, the output is centered at 254 nm. Longer wavelengths may require longer activation time. While some evidence existsthat fluorescent room light can activate the polysorbates, experimentshave shown that use of UV light at 254 nm yields maximal activationbefore room light yields a detectable level of activation.

Air plays an important role in the activation of the polysorbates.Access to air doubles the rate of activation relative to activationsperformed in sealed containers. A shallow reaction chamber with a largesurface area would facilitate oxygen exchange. It is not yet known whichgas is responsible; an oxygen derivative is likely, although peroxidesare not involved. UV exposure of compounds with ether linkages is knownto generate peroxides, which can be detected and quantified usingperoxide test strips. In a reaction, hydrogen peroxide at 1 to 10 foldhigher level than found in UV-activated material was added to apolysorbate solution in the absence of light. No activation wasobtained.

The reagent is placed in a suitable vessel for irradiation. Studies with2% polysorbate 80 indicate that 254 nm light at 1800 μW/cm2 iscompletely absorbed by the solution at a depth of 3-4 cm. Thus, theactivation rate can be maximized by irradiating a relatively thin layer.

As such, a consideration for the vessel is the ability to achieveuniform irradiation. As noted above, a large shallow reaction chamber isdesirable, however, it may be difficult to achieve on a large scale. Tocompensate, simple stirring that facilitates the replenishment of air inthe solution achieves an equivalent result. Thus, if the pathlength islong or the reaction chamber is not shallow, the reagent may be mixed oragitated. The reagent can be activated in any aqueous solution andbuffering is not required.

An exemplary activation takes place in a cuvette with a 1 cm liquidthickness. The reagent is irradiated at a distance of less than 9 cm at1500 μW/cm² (initial source output) for approximately 24 hours. Underthese conditions, the activated reagent converts a minimum of 85% of thepeptide to APO-peptide.

As noted above, the polyoxyalkylenes can be activated via chemicaloxidation with ammonium persulfate. The activation is rapid and theextent of activation increases with the concentration of ammoniumpersulfate. Ammonium persulfate can be used in a range from about0.01%-0.5%, and most preferably from 0.025 to 0.1%. If the levels ofammonium persulfate are too high, the peroxide byproducts can have anadverse effect on the compounds being modified. This adverse effect canbe diminished by treatment of activated polyoxyalkylenes withmercaptoethanol, or another mild reducing agent, which does not inhibitthe formation of APO-therapeutics. Peroxides generated from UV treatmentcan also be reduced by treatment with mercaptoethanol. Furthermore, asnoted above, the UV procedure can be performed in conjunction withchemical activation.

C. Modification of Peptides or Proteins with Activated Reagent

The therapeutics are reacted with the APO reagent in either a liquid orsolid phase and become modified by the attachment of the APO derivative.The methods described herein for attachment offer the advantage ofmaintaining the charge on the therapeutic, such as a peptide or protein.When the charge of the peptide is critical to its function, such as theantibiotic activity of cationic peptides described herein, theseattachment methods offer additional advantages. Methods that attachgroups via acylation result in the loss of positive charge viaconversion of amino to amido groups. In addition, no bulky orpotentially antigenic linker, such as a triazine group, is known to beintroduced by the methods described herein.

As noted above, APO-therapeutic formation occurs in solid phase or inaqueous solution. By way of example, briefly, in the solid phase method,a peptide or other therapeutic is suspended in a suitable buffer, suchas an acetate buffer. Other suitable buffers that supportAPO-therapeutic formation may also be used. The acetate buffer may besodium, potassium, lithium, and the like. Other acetate solutions, suchas HAc or HAc-NaOH, are also suitable. A preferred pH range for thebuffer is from 2 to 8.3, although a wider range may be used. When thestarting pH of the acetic acid-NaOH buffer is varied, subsequentlyophilization from 200 mM acetic acid buffer yields only the Type Imodified peptide (see Example 14). The presence of an alkaline buffercomponent results in the formation of Type II modified peptides. Atypical peptide concentration is 1 mg/ml, which results in 85-95%modified peptide, however other concentrations are suitable. The majorconsideration for determining concentration appears to be economic. Theactivated polymer (APO) is added in molar excess to the therapeutic.Generally, a starting ratio of approximately 2.5:1 (APO:therapeutic) to5:1 (APO:therapeutic) generates APO-modified therapeutic in good yield.

The reaction mix is then frozen (e.g., −80° C.) and lyophilized. Sodiumacetate disproportionates into acetic acid and NaOH duringlyophilization; removal of the volatile acetic acid by the vacuum leavesNaOH dispersed throughout the result solid matrix. This loss of aceticacid is confirmed by a pH increase detected upon dissolution of thelyophilizate. No APO-modified therapeutic is formed in acetate buffer ifthe samples are only frozen then thawed.

The modification reaction can also take place in aqueous solution.However, APO modifications do not occur at ambient temperature in anyacetate buffer system tested regardless of pH. APO modifications alsoare not formed in phosphate buffers as high as pH 11.5. APO modificationdoes occur in a sodium carbonate buffer at a pH greater than about 8.5.Other buffers may also be used if they support derivatization. A pHrange of 9-11 is also suitable, and pH 10 is most commonly used. Thereaction occurs in two phases: Type I modified peptides form first,followed by formation of Type II modified peptides.

In the present invention, linkage occurs at an amino or a nucleophilicgroup. The amino group may be a primary amine, a secondary amine, or anaryl amine. Nucleophilic groups that may be APO-modified include, butare not limited to, hydrazine derivatives, hydroxylamine derivatives,and sulfhydryl compounds. Preferably, the modification occurs at anamino group, more preferably at a primary or secondary amino group, andmost preferably at a primary amino group.

For a peptide, linkage can occur at the α-NH₂ of the N-terminal aminoacid or ε-NH₂ group of lysine. Other primary and secondary amines mayalso be modified. Complete blocking of all amino groups by acylation(MBI 11CNY1) inhibits APO-peptide formation. Thus, modification ofarginine or tryptophan residues does not occur. If the only amino groupavailable is the α-amino group (e.g., MBI 11B9CN and MBI 11G14CN), theType I form is observed. The inclusion of a single lysine (e.g., MBI11B1CN, MBI 11B7CN, MBI 11B8CN), providing an ε-amino group, results inType II forms as well. The amount of Type II formed increases forpeptides with more lysine residues.

Many antibiotics have free amine groups. Such antibiotics include butare not limited to ampicillin, amoxicillin, amikacin, ciprofloxacin,gentamicin, teicoplanin, tobramycin, and vancomycin. Using the methodsdescribed herein, several peptides, including indolicidin, indolicidinanalogues, gramicidin and bacitracin-2 have been polymer modified.

Examples of compounds that have modified by the solid phase method arelisted in the table below.

TABLE 7 Compound Action Modification Amoxicillin penicillin antibioticYes Amphotericin B anti-fungal No Ampicillin penicillin antibiotic YesBacitracin peptide antibiotic Yes Cephalosporin C aminoglycosideantibiotic No Ciprofloxacin quinolone antibiotic Uncertain*4,4′-Diaminodiphenyl Sulfone anti-leprotic Yes Gentamicin aminoglycosideantibiotic Yes Gramicidin S peptide antibiotic Yes Sulfadiazinesulfonamide antibiotic No Vancomycin glycopeptide antibiotic Yes*Ciprofloxacin was partially destroyed by the process.

D. Purification and Physical Properties of APO-Modified Therapeutics

The APO-modified therapeutics may be purified. In circumstances in whichthe free therapeutic, such as a peptide is toxic, purification may benecessary to remove unmodified therapeutic and/or unreactedpolyoxyalkylenes. Any of a variety of purification methods may be used.Such methods include reversed phase HPLC, precipitation by organicsolvent to remove polysorbate, size exclusion chromatography, ionexchange chromatography, filtration and the like. RP-HPLC is preferred.Procedures for these separation methods are well known.

APO-therapeutic formation can result in the generation of products thatare more hydrophobic than the parent compound. This property can beexploited to effect separation of the conjugate from free compound byRP-HPLC. As shown herein, peptide-conjugates are resolved into twopopulations based on their hydrophobicity as determined by RP-HPLC; theType I population elutes slightly earlier than the Type II population.

The MBI 11 series of peptides have molecular weights between 1600 and2500. When run on a Superose 12 column, a size exclusion column, thesepeptides adsorb to the resin, giving long retention times. In contrast,the APO-modified peptides do not adsorb and elute at 50 kDa(MBI11CN-Tw80) and at 69 kDa (MBI 11A3CN-Tw80), thus demonstrating alarge increase in apparent molecular mass (Stokes radius).

An increase in apparent molecular mass could enhance thepharmacokinetics of peptides in particular because increased molecularmass reduces the rate at which peptides and proteins are removed fromblood. Micelle formation may offer additional benefits by delivering“packets” of peptide molecules to microorganisms rather than relying onthe multiple binding of single peptide molecules. In addition,APO-modified peptides are soluble in methylene chloride or chloroform(e.g., to at least 10 mg/mL), whereas the parent peptide is essentiallyinsoluble. This increased organic solubility may significantly enhancethe ability to penetrate tissue barriers and may be exploited for asimplified purification of the APO-peptide. The increased solubility inorganic media may also allow the formulation of peptides in oil or lipidbased delivery systems which target specific sites, such as solidtumors.

In addition, by circular dichroism (CD) studies, APO-modified peptidesare observed to have an altered 3-dimensional conformation. As shown inthe Examples, MBI 11CN and MBI 11B7CN have unordered structures inphosphate buffer or 40% aqueous trifluoroethanol (TFE) and form a β-turnconformation only upon insertion into liposomes. In contrast, CD spectrafor APO-modified MBI 11CN and APO-modified MBI 11B7CN indicate β-turnstructure in phosphate buffer.

Cationic peptides appear to maintain their original charge aftermodification with an APO, thereby preventing loss of activity sometimescaused by acylation reactions. Moreover, the present methods are notknown to introduce antigenic linkers.

E. Biological Properties of APO-Modified Therapeutics

The biological properties of APO-modified therapeutics appear to beimproved compared to unmodified therapeutics. For example, modified andunmodified peptides are compared. Because the product consists of apeptide of known composition coupled to one or more polyoxyalkylenecomponents derived from a polymeric mixture, defining an exact molecularweight for concentration calculations is not readily achieved. It ispossible, however, to determine the concentration by spectrophotometricassay. Such a measurement is used to normalize APO-peptideconcentrations for biological assays. For example, a 1 mg/mLMBI11CN-Tw80 solution contains the same amount of cationic peptide as a1 mg/mL solution of the parent peptide, thus allowing direct comparisonof toxicity and efficacy data. The modified peptides have an equivalentMIC to unmodified peptides. In vivo, however, the modified peptidesdemonstrate a lower LC₅₀ than the unmodified peptides against a panel oftumor cell lines. Thus, formation of APO-peptides increases the potencyof cationic peptides against cancer cells in culture.

In general, the efficacy of a modified therapeutic is determined by invitro and in vivo assays used for the unmodified therapeutic. Thus, theassays employed depend upon the therapeutic. Assays for the therapeuticsdisclosed herein are well known. Assays include those for biologicalactivity, pharmacokinetics, toxicity, adverse reactions, immunogenicity,and the like. Such assays are available to those skilled in the art.

VII. Formulations and Administration

As noted above, the present invention provides methods for treating andpreventing infections by administering to a patient a therapeuticallyeffective amount of a peptide analogue of indolicidin as describedherein. Patients suitable for such treatment may be identified bywell-established hallmarks of an infection, such as fever, pus, cultureof organisms, and the like. Infections that may be treated with peptideanalogues include those caused by or due to microorganisms. Examples ofmicroorganisms include bacteria (e.g., Gram-positive, Gram-negative),fungi, (e.g., yeast and molds), parasites (e.g., protozoans, nematodes,cestodes and trematodes), viruses, and prions. Specific organisms inthese classes are well known (see for example, Davis et al.,Microbiology, 3^(rd) edition, Harper & Row, 1980). Infections include,but are not limited to, toxic shock syndrome, diphtheria, cholera,typhus, meningitis, whooping cough, botulism, tetanus, pyogenicinfections, dysentery, gastroenteritis, anthrax, Lyme disease, syphilis,rubella, septicemia and plague.

More specifically, clinical indications include, but are not limited to:1/infections following insertion of intravascular devices or peritonealdialysis catheters; 2/infection associated with medical devices orprostheses; 3/infection during hemodialysis; 4/S. aureus nasal andextra-nasal carriage; 5/burn wound infections; 6/surgical wounds,7/acne, including severe acne vulgaris; 8/nosocomial pneumonia;9/meningitis; 10/cystic fibrosis; 11/infective endocarditis;12/osteomyelitis; and 13/sepsis in an immunocompromised host.

1/Infections following insertion of contaminated intravascular devices,such as central venous catheters, or peritoneal dialysis catheters.These catheters are cuffed or non-cuffed, although the infection rate ishigher for non-cuffed catheters. Both local and systemic infection mayresult from contaminated intravascular devices, more than 25,000patients develop device related bacteremia in the United States eachyear. The main organisms responsible are coagulase-negativestaphylococci (CoNS), Staphylococcus aureus, Enterococcus spp, E. coliand Candida spp.

The peptide and/or antibiotic, preferably as an ointment or cream, canbe applied to the catheter site prior to insertion of the catheter andthen again at each dressing change. The peptide may be incorporated intothe ointment or cream at a concentration preferably of about 0.5 toabout 2% (w/v).

2/Infection associated with medical devices or prostheses, e.g.catheter, grafts, prosthetic heart valves, artificial joints, etc. Oneto five percent of indwelling prostheses become infected which usuallyrequires removal or replacement of the prostheses. The main organismsresponsible for these infections are CoNS and S. aureus.

Preferably, the peptide and/or antibiotic can be coated, eithercovalently bonded or by any other means, onto the medical device eitherat manufacture of the device or after manufacture but prior to insertionof the device. In such an application, the peptide antibiotic ispreferably applied as a 0.5 to 2% solution.

3/Infection during hemodialysis. Infection is the second leading causeof death in patients on chronic hemodialysis. Approximately 23% ofbacteremias are due to access site infections. The majority of graftinfections are caused by coagulate-positive (S. aureus) andcoagulate-negative staphylococci. To combat infection, the peptide aloneor in combination with an antibiotic can be applied as an ointment orcream to the dialysis site prior to each hemodialysis procedure.

4/S. aureus nasal and extra-nasal carriage. Infection by this organismmay result in impetigenous lesions or infected wounds. It is alsoassociated with increased infection rates following cardiac surgery,hemodialysis, orthopedic surgery and neutropenia, both disease inducedand iatrogenic. Nasal and extra-nasal carriage of staphylococci canresult in hospital outbreaks of the same staphylococci strain that iscolonizing a patient's or hospital worker's nasal passage or extra-nasalsite. Much attention has been paid to the eradication of nasalcolonization, but the results of treatment have been generallyunsatisfactory. The use of topical antimicrobial substances, such asBacitracin, Tetracycline, or Chlorhexidine, results in the suppressionof nasal colonization, as opposed to its eradication.

The peptide alone or in combination with an antibiotic are preferablyapplied intra-nasally, formulated for nasal application, as a 0.5 to 2%ointment, cream or solution. Application may occur once or multipletimes until the colonization of staphylococci is reduced or eliminated.

5/Burn wound infections. Although the occurrence of invasive burn woundinfections has been significantly reduced, infection remains the mostcommon cause of morbidity and mortality in extensively burned patients.Infection is the predominant determinant of wound healing, incidence ofcomplications, and outcome of burn patients. The main organismsresponsible are Pseudomonas aeruginosa, S. aureus, Streptococcuspyogenes, and various gram-negative organisms. Frequent debridements andestablishment of an epidermis, or a surrogate such as a graft or a skinsubstitute, is essential for prevention of infection.

The peptide alone or in combination with antibiotics can be applied toburn wounds as an ointment or cream and/or administered systemically.Topical application may prevent systemic infection following superficialcolonization or eradicate a superficial infection. The peptide ispreferably administered as a 0.5 to 2% cream or ointment. Application tothe skin could be done once a day or as often as dressings are changed.The systemic administration could be by intravenous, intramuscular orsubcutaneous injections or infusions. Other routes of administrationcould also be used.

6/Surgical wounds, especially those associated with foreign material,e.g. sutures. As many as 71% of all nosocomial infections occur insurgical patients, 40% of which are infections at the operative site.Despite efforts to prevent infection, it is estimated that between500,000 and 920,000 surgical wound infections complicate theapproximately 23 million surgical procedures performed annually in theUnited States. The infecting organisms are varied but staphylococci areimportant organisms in these infections.

The peptide alone or with an antibiotic may be applied as an ointment,cream or liquid to the wound site or as a liquid in the wound prior toand during closure of the wound. Following closure the peptideantibiotic could be applied at dressing changes. For wounds that areinfected, the peptide antibiotic could be applied topically and/orsystemically.

7/Acne, including severe acne vulgaris. This condition is due tocolonization and infection of hair follicles and sebaceous cysts byPropionibacterium acne. Most cases remain mild and do not lead toscarring although a subset of patients develop large inflammatory cystsand nodules, which may drain and result in significant scarring.

The peptide alone or with an antibiotic can be incorporated into soap orapplied topically as a cream, lotion or gel to the affected areas eitheronce a day or multiple times during the day. The length of treatment maybe for as long as the lesions are present or used to prevent recurrentlesions. The peptide antibiotic could also be administered orally orsystemically to treat or prevent acne lesions.

8/Nosocomial pneumonia. Nosocomial pneumonias account for nearly 20% ofall nosocomial infections. Patients most at risk for developingnosocomial pneumonia are those in an intensive care units, patients withaltered levels of consciousness, elderly patients, patients with chroniclung disease, ventilated patients, smokers and post-operative patients.In a severely compromised patient, multiantibiotic-resistant nosocomialpathogens are likely to be the cause of the pneumonia.

The main organisms responsible are P. aeruginosa, S. aureus, Klebsiellapneumoniae and Enterobacter spp. The peptide alone or in combinationwith other antibiotics could be administered orally or systemically totreat pneumonia. Administration could be once a day or multipleadministrations per day. Peptide antibiotics could be administereddirectly into the lung via inhalation or via installation of anendotracheal tube.

9/Meningitis. Bacterial meningitis remains a common disease worldwide.Approximately 25,000 cases occur annually, of which 70% occur inchildren under 5 years of age. Despite an apparent recent decline in theincidence of severe neurologic sequelae among children survivingbacterial meningitis, the public health problems as a result of thisdisease are significant worldwide. The main responsible organisms are H.influenzae, Streptococcus pneumoniae and Neisseria meningitidis.Community acquired drug resistant S. pneumoniae are emerging as awidespread problem in the United States. The peptide alone or incombination with known antibiotics could be administered orally orsystemically to treat meningitis. The preferred route would beintravenously either once a day or multiple administration per day.Treatment would preferably last for up to 14 days.

10/Cystic fibrosis. Cystic fibrosis (CF) is the most common geneticdisorder of the Caucasian population. Pulmonary disease is the mostcommon cause of premature death in cystic fibrosis patients. Optimumantimicrobial therapy for CF is not known, and it is generally believedthat the introduction of better anti-pseudomonal antibiotics has beenthe major factor contributing to the increase in life expectancy for CFpatients. The most common organisms associated with lung disease in CFare S. aureus, P. aeruginosa and H. influenzae.

The peptide alone or in combination with other antibiotics could beadministrated orally or systemically or via aerosol to treat cysticfibrosis. Preferably, treatment is effected for up to 3 weeks duringacute pulmonary disease and/or for up to 2 weeks every 2-6 months toprevent acute exacerbations.

11/Infective endocarditis. Infective endocarditis results from infectionof the heart valve cusps, although any part of the endocardium or anyprosthetic material inserted into the heart may be involved. It isusually fatal if untreated. Most infections are nosocomial in origin,caused by pathogens increasingly resistant to available drugs. The mainorganisms responsible are Viridans streptococci, Enterococcus spp, S.aureus and CoNS.

The peptide alone or in combination with other antibiotics could beadministered orally or systemically to treat endocarditis, althoughsystemic administration would be preferred. Treatment is preferably for2-6 weeks in duration and may be given as a continuous infusion ormultiple administration during the day.

12/Osteomyelitis. In early acute disease the vascular supply to the boneis compromised by infection extending into surrounding tissue. Withinthis necrotic and ischemic tissue, the bacteria may be difficult toeradicate even after an intense host response, surgery, and/orantibiotic therapy. The main organisms responsible are S. aureus, E.coli, and P. aeruginosa.

The peptide antibiotic could be administered systemically alone or incombination with other antibiotics. Treatment would be 2-6 weeks induration. The peptide antibiotic could be given as a continuous infusionor multiple administration during the day. Peptide antibiotic could beused as an antibiotic-impregnated cement or as antibiotic coated beadsfor joint replacement procedures.

13/Sepsis in immunocompromised host. Treatment of infections in patientswho are immunocompromised by virtue of chemotherapy-inducedgranulocytopenia and immunosuppression related to organ or bone marrowtransplantation is always a big challenge. The neutropenic patient isespecially susceptible to bacterial infection, so antibiotic therapyshould be initiated promptly to cover likely pathogens, if infection issuspected. Organisms likely to cause infections in granulocytopenicpatients are: S. epidermidis, S. aureus, S. viridans, Enterococcus spp,E. coli, Klebsiella spp, P. aeruginosa and Candida spp.

The peptide alone or with an antibiotic is preferably administeredorally or systemically for 2-6 weeks in duration. The peptide antibioticcould be given as a continuous infusion or multiple administrationduring the day.

Effective treatment of infection may be examined in several differentways. The patient may exhibit reduced fever, reduced number oforganisms, lower level of inflammatory molecules (e.g., IFN-γ, IL-12,IL-1, TNF), and the like.

The in vivo therapeutic efficacy from administering a cationic peptideand antibiotic agent in combination is based on a successful clinicaloutcome and does not require 100% elimination of the organisms involvedin the infection. Achieving a level of antimicrobial activity at thesite of infection that allows the host to survive or eradicate themicroorganism is sufficient. When host defenses are maximally effective,such as in an otherwise healthy individual, only a minimal antimicrobialeffect may suffice. Thus, reducing the organism load by even one log (afactor of 10) may permit the defenses of the host to control theinfection. In addition, clinical therapeutic success may depend more onaugmenting an early bactericidal effect than on the long-term effect.These early events are a significant and critical part of therapeuticsuccess, because they allow time for the host defense mechanisms toactivate. This is especially true for life-threatening infections (e.g.meningitis) and other serious chronic infections (e.g. infectiveendocarditis).

Peptides and antibiotic agents of the present invention are preferablyadministered as a pharmaceutical composition. Briefly, pharmaceuticalcompositions of the present invention may comprise one or more of thepeptide analogues described herein, in combination with one or morephysiologically acceptable carriers, diluents, or excipients. As notedherein, the formulation buffer used may affect the efficacy or activityof the peptide analogue. A suitable formulation buffer contains bufferand solubilizer. The formulation buffer may comprise buffers such assodium acetate, sodium citrate, neutral buffered saline,phosphate-buffered saline, and the like or salts, such as NaCl. Sodiumacetate is preferred. In general, an acetate buffer from 5 to 500 mM isused, and preferably from 100 to 200 mM. The pH of the final formulationmay range from 3 to 10, and is preferably approximately neutral (aboutpH 7-8). Solubilizers, such as polyoxyethylenesorbitans (e.g., Tween 80,Tween 20) and polyoxyethylene ethers (e.g., Brij 56) may also be addedif the compound is not already polymer-modified.

Although the formulation buffer is exemplified herein with peptideanalogues of the present invention, this buffer is generally useful anddesirable for delivery of other peptides. Peptides that may be deliveredin this formulation buffer include indolicidin, other indolicidinanalogues (see, PCT WO 95/22338), bacteriocins, gramicidin, bactenecin,drosocin, polyphemusins, defensins, cecropins, melittins,cecropin/melittin hybrids, magainins, sapecins, apidaecins, protegrins,tachyplesins, thionins; IL-1 through 15; corticotropin-releasinghormone; human growth hormone; insulin; erythropoietin; thrombopoietin;myelin basic protein peptides; various colony stimulating factors suchas M-CSF, GM-CSF, kit ligand; and peptides and analogues of these andsimilar proteins.

Additional compounds may be included in the compositions. These include,for example, carbohydrates such as glucose, mannose, sucrose ordextrose, mannitol, other proteins, polypeptides or amino acids,chelating agents such as EDTA or glutathione, adjuvants andpreservatives. As noted herein, pharmaceutical compositions of thepresent invention may also contain one or more additional activeingredients, such as an antibiotic (see discussion herein on synergy) orcytokine.

The compositions may be administered in a delivery vehicle. For example,the composition can be encapsulated in a liposome (see, e.g., WO96/10585; WO 95/35094), complexed with lipids, encapsulated inslow-release or sustained release vehicles, such as poly-galactide, andthe like. Within other embodiments, compositions may be prepared as alyophilizate, utilizing appropriate excipients to provide stability.

Pharmaceutical compositions of the present invention may be administeredin various manners. For example, cationic peptides with or withoutantibiotic agents may be administered by intravenous injection,intraperitoneal injection or implantation, subcutaneous injection orimplantation, intradermal injection, lavage, inhalation, implantation,intramuscular injection or implantation, intrathecal injection, bladderwash-out, suppositories, pessaries, topical (e.g., creams, ointments,skin patches, eye drops, ear drops, shampoos) application, enteric,oral, or nasal route. The combination is preferably administeredintravenously. Systemic routes include intravenous, intramuscular orsubcutaneous injection (including a depot for long-term release),intraocular or retrobulbar, intrathecal, intraperitoneal (e.g. byintraperitoneal lavage), transpulmonary using aerosolized or nebulizeddrug or transdermal. Topical routes include administration in the formof salves, ophthalmic drops, ear drops, or irrigation fluids (for, e.g.irrigation of wounds). The compositions may be applied locally as aninjection, drops, spray, tablets, cream, ointment, gel, and the like.They may be administered as a bolus or as multiple doses over a periodof time.

The level of peptide in serum and other tissues after administration canbe monitored by various well-established techniques such as bacterial,chromatographic or antibody based, such as ELISA, assays.

Pharmaceutical compositions of the present invention are administered ina manner appropriate to the infection or disease to be treated. Theamount and frequency of administration will be determined by factorssuch as the condition of the patient, the cause of the infection, andthe severity of the infection. Appropriate dosages may be determined byclinical trials, but will generally range from about 0.1 to 50 mg/kg.The general range of dosages for the antibiotic agents are presentedbelow.

TABLE 8 ANTIMICROBIAL AGENT DOSE RANGE Ciprofloxacin 400-1500 mg/dayGentamicin 3 mg/kg/day Tobramycin 3 mg/kg/day Imipenem 1500 mg/kg every12 h Piperacillin 24 g/day Vancomycin, Teicoplanin 6-30 mg/kg/dayStreptomycin 500 mg-1 g/every 12 h Methicillin 100-300 mg/dayAmpicillin, Amoxicillin 250-500 mg/every 8 h Penicillin 200,000units/day Ceftriaxone 4 g/day Cefotaxime 12 g/day Metronidazole 4 g/dayTetracycline 500 mg/every 6 h Rifampin 600 mg/day Fluconazole 150-400mg/day Acyclovir 200-400 mg/day Ribavirin 20 mg/ml (aerosol).Amantadine-rimantadine 200 mg/day Metronidazole 2 g/day Cotrimoxazole15-20 mg/kg/day Chloroquine 800 mg/day

In addition, the compositions of the present invention may be used inthe manner of common disinfectants or in any situation in whichmicroorganisms are undesirable. For example, these peptides may be usedas surface disinfectants, coatings, including covalent bonding, formedical devices, coatings for clothing, such as to inhibit growth ofbacteria or repel mosquitoes, in filters for air purification, such ason an airplane, in water purification, constituents of shampoos andsoaps, food preservatives, cosmetic preservatives, media preservatives,herbicide or insecticides, constituents of building materials, such asin silicone sealant, and in animal product processing, such as curing ofanimal hides. As used herein, “medical device” refers to any device foruse in a patient, such as an implant or prosthesis. Such devicesinclude, stents, tubing, probes, cannulas, catheters, synthetic vasculargrafts, blood monitoring devices, artificial heart valves, needles, andthe like.

For these purposes, typically the peptides alone or in conjunction withan antibiotic are included in compositions commonly employed or in asuitable applicator, such as for applying to clothing. They may beincorporated or impregnated into the material during manufacture, suchas for an air filter, or otherwise applied to devices. The peptides andantibiotics need only be suspended in a solution appropriate for thedevice or article. Polymers are one type of carrier that can be used.

The peptides, especially the labeled analogues, may be used in imageanalysis and diagnostic assays or for targeting sites in eukaryoticmulticellular and single cell cellular organisms and in prokaryotes. Asa targeting system, the analogues may be coupled with other peptides,proteins, nucleic acids, antibodies and the like.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Synthesis Purification and Characterization ofCationic Peptides and Analogues

Peptide synthesis is based on the standard solid-phase Fmoc protectionstrategy. The instrument employed is a 9050 Plus PepSynthesiser(PerSeptive BioSystems Inc.). Polyethylene glycol polystyrene (PEG-PS)graft resins are employed as the solid phase, derivatized with anFmoc-protected amino acid linker for C-terminal amide synthesis. HATU(4-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) is used as the coupling reagent. During synthesis,coupling steps are continuously monitored to ensure that each amino acidis incorporated in high yield. The peptide is cleaved from thesolid-phase resin using trifluoroacetic acid and appropriate scavengersand the crude peptide is purified using preparative reversed-phasechromatography. Typically the peptide is prepared as thetrifluoroacetate salt, but other salts, such as acetate, chloride andsulfate, can also be prepared by salt exchange.

All peptides are analyzed by mass spectrometry to ensure that theproduct has the expected molecular mass. The product should have asingle peak accounting for >95% of the total peak area when subjected toanalytical reversed-phase high performance liquid chromatography(RP-HPLC), a separation method that depends on the hydrophobicity of thepeptide. In addition, the peptide should show a single band accountingfor >90% of the total band intensity when subjected to acid-urea gelelectrophoresis, a separation method based on the charge to mass rationof the peptide.

Peptide content, the amount of the product that is peptide rather thanretained water, salt or solvent, is measured by quantitative amino acidanalysis, free amine derivatization or spectrophotometric quantitation.Amino acid analysis also provides information on the ratio of aminoacids present in the peptide, which assists in confirming theauthenticity of the peptide.

Peptide analogues and their names are listed below. In this list, andelsewhere, the amino acids are denoted by the one-letter amino acid codeand lower case letters represent the D-form of the amino acid.

Apidaecin IA (SEQ ID NO: 96) G N N R P V Y I P Q P R P P H P R IDeber A2KA2 (SEQ ID NO: 97)K K A A A K A A A A A K A A W A A K A A A K K K K 10 (SEQ ID NO: 98)I L P W K W P W W P W R R 10CN (SEQ ID NO: 98) I L P W K W P W W P W R R11 (SEQ ID NO: 99) I L K K W P W W P W R R K 11CN (SEQ ID NO: 99)I L K K W P W W P W R R K 11CNR (SEQ ID NO: 25)K R R W P W W P W K K L I 11A1CN (SEQ ID NO: 100)I L K K F P F F P F R R K 11A2CN (SEQ ID NO: 33)I L K K I P I I P I R R K 11A3CN (SEQ ID NO: 34)I L K K Y P Y Y P Y R R K 11A4CN (SEQ ID NO: 65) I L K K W P W P W R R K11A5CN (SEQ ID NO: 35) I L K K Y P W Y P W R R K 11A6CN (SEQ ID NO: 36)I L K K F P W F P W R R K 11A7CN (SEQ ID NO: 37)I L K K F P F W P W R R K 11A8CN (SEQ ID NO: 38) I L R Y V Y Y V Y R R K11A9CN (SEQ ID NO: 39) I L R W P W W P W W P W R R K 11A10CN(SEQ ID NO: 40) W W R W P W W P W R R K 11B1CN (SEQ ID NO: 41)I L R R W P W W P W R R K 11B2CN (SEQ ID NO: 42) I L R R W P W W P W R K11B3CN (SEQ ID NO: 43) I L K W P W W P W R R K 11B4CN (SEQ ID NO: 44)I L K K W P W W P W R K 11B5CN (SEQ ID NO: 45) I L K W P W W P W R K11B7CN (SEQ ID NO: 101) I L R W P W W P W R R K 11B7CNR (SEQ ID NO: 46)K R R W P W W P W R L I 11B8CN (SEQ ID NO: 66) I L W P W W P W R R K11B9CN (SEQ ID NO: 102) I L R R W P W W P W R R R 11B10CN(SEQ ID NO: 103) I L K K W P W W P W K K K 11B16CN (SEQ ID NO: 47)I L R W P W W P W R R K I M I L K K A G S 11B17CN (SEQ ID NO: 24)I L R W P W W P W R R K M I L K K A G S 11B18CN (SEQ ID NO: 48)I L R W P W W P W R R K D M I L K K A G S 11B19CN (SEQ ID NO: 49)I L R W P W R R W P W R R K 11B20CN (SEQ ID NO: 104)I L R W P W W P W R R K I L M R W P W W P W R R K M A A 11C3CN(SEQ ID NO: 105) I L K K W A W W P W R R K 11C4CN (SEQ ID NO: 106)I L K K W P W W A W R R K 11C5CN (SEQ ID NO: 107)W W K K W P W W P W R R K 11D1CN (SEQ ID NO: 67) L K K W P W W P W R R K11D3CN (SEQ ID NO: 68) P W W P W R R K 11D4CN (SEQ ID NO: 69)I L K K W P W W P W R R K M I L K K A G S 11D5CN (SEQ ID NO: 51)I L K K W P W W P W R R M I L K K A G S 11D6CN (SEQ ID NO: 52)I L K K W P W W P W R R I M I L K K A G S 11D9M8 (SEQ ID NO: 70)W W P W R R K 11D10M8 (SEQ ID NO: 71) I L K K W P W 11D11H(SEQ ID NO: 108) I L K K W P W W P W R R K M 11D12H (SEQ ID NO: 109)I L K K W P W W P W R R M 11D13H (SEQ ID NO: 110)I L K K W P W W P W R R I M 11D14CN (SEQ ID NO: 55)I L K K W W W P W R K 11D15CN (SEQ ID NO: 56) I L K K W P W W W R K11D18CN (SEQ ID NO: 26) W R I W K P K W R L P K W 11D19CN(SEQ ID NO: 111) C L R W P W W P W R R K 11E1CN (SEQ ID NO: 99)i L K K W P W W P W R R K 11E2CN (SEQ ID NO: 99)I L K K W P W W P W R R k 11E3CN (SEQ ID NO: 99)i L K K W P W W P W R R k 11F1CN (SEQ ID NO: 57)I L K K W V W W V W R R K 11F2CN (SEQ ID NO: 58)I L K K W P W W V W R R K 11F3CN (SEQ ID NO: 59)I L K K W V W W P W R R K 11F4CN (SEQ ID NO: 27) I L R W V W W V W R R K11F4CNR (SEQ ID NO: 66) K R R W V W W V W R L I 11F5CN (SEQ ID NO: 28)I L R R W V W W V W R R K 11F6CN (SEQ ID NO: 61)I L R W W V W W V W W R R K 11G2CN (SEQ ID NO: 73)I K K W P W W P W R R K 11G3CN (SEQ ID NO: 74) I L K K P W W P W R R K11G4CN (SEQ ID NO: 75) I L K K W W W P W R R K 11G5CN (SEQ ID NO: 76)I L K K W P W W W R R K 11G6CN (SEQ ID NO: 77) I L K K W P W W P R R K11G7CN (SEQ ID NO: 112) I L K K W P W W P W R R 11G13CN (SEQ ID NO: 113)I L K K W P W W P W K 11G14CN (SEQ ID NO: 114) I L K K W P W W P W R11G24CN (SEQ ID NO: 81) L W P W W P W R R K 11G25CN (SEQ ID NO: 29)L R W W W P W R R K 11G26CN (SEQ ID NO: 62) L R W P W W P W 11G27CN(SEQ ID NO: 80) W P W W P W R R K 11G28CN (SEQ ID NO: 63)R W W W P W R R K 11H1CN (SEQ ID NO: 30) A L R W P W W P W R R K 11H2CN(SEQ ID NO: 82) I A R W P W W P W R R K 11H3CN (SEQ ID NO: 83)I L A W P W W P W R R K 11H4CN (SEQ ID NO: 84) I L R A P W W P W R R K11H5CN (SEQ ID NO: 31) I L R W A W W P W R R K 11H6CN (SEQ ID NO: 85)I L R W P A W P W R R K 11H7CN (SEQ ID NO: 86) I L R W P W A P W R R K11H8CN (SEQ ID NO: 87) I L R W P W W A W R R K 11H9CN (SEQ ID NO: 88)I L R W P W W P A R R K 11H10CN (SEQ ID NO: 89) I L R W P W W P W A R K11H11CN (SEQ ID NO: 90) I L R W P W W P W R A K 11H12CN (SEQ ID NO: 91)I L R W P W W P W R R A 11J01CN (SEQ ID NO: 64)R R I W K P K W R L P K R 11J02CN (SEQ ID NO: 32)W R W W K P K W R W P K W 21A1 (SEQ ID NO: 115)K K W W R R V L S G L K T A G P A I Q S V L N K 21A2 (SEQ ID NO: 116)K K W W R R A L Q G L K T A G P A I Q S V L N K 21A10 (SEQ ID NO: 117)K K W W R R V L K G L S S G P A L S N V 22A1 (SEQ ID NO: 118)K K W W R R A L Q A L K N G L P A L I S 26 (SEQ ID NO: 119)K W K S F I K K L T S A A K K V V T T A K P L I S S 27 (SEQ ID NO: 120)K W K L F K K I G I G A V L K V L T T G L P A L I S 28 (SEQ ID NO: 121)K W K L F K K I G I G A V L K V L T T G L P A L K L T K 29(SEQ ID NO: 122) K W K S F I K K L T T A V K K V L T T G L P A L I S29A2 (SEQ ID NO: 123)K W K S F I K N L T K V L K K V V T T A L P A L I S 29A3(SEQ ID NO: 124) K W K S F I K K L T S A A K K V L T T G L P A L I S29F1 (SEQ ID NO: 125)K W K L F I K K L T P A V K K V L L T G L P A L I S 31 (SEQ ID NO: 126)G K P R P Y S P I P T S P R P I R Y REWH 53A5 (SEQ ID NO: 127)R L A R I V V I R V A R CN suffix = amidated C-terminus H suffix =homoserine at C-terminus M suffix = MAP branched peptide R suffix =retro-synthesized peptide

Example 2 Synthesis of Modified Peptides

Cationic peptides, such as indolicidin analogues, are modified to alterthe physical properties of the original peptide, either by use ofmodified amino acids in synthesis or by post-synthetic modification.Such modifications include: acetylation at the N-terminus,Fmoc-derivatized N-terminus, polymethylation, peracetylation, andbranched derivatives.

α-N-terminal acetylation. Prior to cleaving the peptide from the resinand deprotecting it, the fully protected peptide is treated withN-acetylimidazole in DMF for 1 hour at room temperature, which resultsin selective reaction at the α-N-terminus. The peptide is thendeprotected/cleaved and purified as for an unmodified peptide.

Fmoc-derivatized α-N-terminus. If the final Fmoc deprotection step isnot carried out, the α-N-terminus Fmoc group remains on the peptide. Thepeptide is then side-chain deprotected/cleaved and purified as for anunmodified peptide.

Polymethylation. The purified peptide in a methanol solution is treatedwith excess sodium bicarbonate, followed by excess methyl iodide. Thereaction mixture is stirred overnight at room temperature, extractedwith organic solvent, neutralized and purified as for an unmodifiedpeptide. Using this procedure, a peptide is not fully methylated;methylation of MBI 11CN yielded an average of 6 methyl groups. Thus, themodified peptide is a mixture of methylated products.

Peracetylation. A purified peptide in DMF solution is treated withN-acetylimidazole for 1 hour at room temperature. The crude product isconcentrated, dissolved in water, lyophilized, re-dissolved in water andpurified as for an unmodified peptide. Complete acetylation of primaryamine groups is observed.

Four/eight branch derivatives. The branched peptides are synthesized ona four or eight branched core bound to the resin. Synthesis anddeprotection/cleavage proceed as for an unmodified peptide. Thesepeptides are purified by dialysis against 4 M guanidine hydrochloridethen water, and analyzed by mass spectrometry.

Peptides modified using the above procedures are listed in Table 9.

TABLE 9 Peptide modified Peptide name Sequence Modification 10 10AI L P W K W P W W P W R R Acetylated α-N-terminus (SEQ ID NO: 98) 11 11AI L K K W P W W P W R R K Acetylated α-N-terminus (SEQ ID NO: 99) 11CN11ACN I LK K W P W W P W R R K Acetylated α-N-terminus (SEQ ID NO: 99)11CN 11CNW1 I L K K W P W W P W R R K Fmoc-derivatized N-terminus(SEQ ID NO: 99) 11CN 11CNX1 I L K K W P W W P W R R KPolymethylated derivative (SEQ ID NO: 99) 11CN 11CNY1I L K K W P W W P W R R K Peracetylated derivative (SEQ ID NO: 99) 1111M4 I L K K W P W W P W R R K Four branch derivative (SEQ ID NO: 98) 1111M8 I L K K W P W W P W R R K Eight branch derivative (SEQ ID NO: 98)11B1CN 11B1CNW1 I L R R W P W W P W R R K Fmoc-derivatized N-terminus(SEQ ID NO: 41) 11B4CN 11B4ACN I L K K W P W W P W R KAcetylated N-terminus (SEQ ID NO: 44) 11B7CN 11B7ACNI L R W P W W P W R R K Acetylated N-terminus (SEQ ID NO: 101) 11B7CN11B7CNF12 I L R W P W W P W R R K Formylated Lys[12] (SEQ ID NO: 101)11B9CN 11B9ACN I L R R W P W W P W R R R Acetylated N-terminus(SEQ ID NO: 102) 11D9 11D9M8 W W P W R R K Eight branch derivative(SEQ ID NO: 70) 11D10 11D10M8 I L K K W P W Eight branch derivative(SEQ ID NO: 71) 11G6CN 11G6ACN I L K K W P W W P R R KAcetylated α-N-terminus (SEQ ID NO: 77) 11G7CN 11G7ACNI L K K W P W W P W R R Acetylated α-N-terminus (SEQ ID NO: 112)

Example 3 Recombinant Production of Peptide Analogues

Peptide analogues are alternatively produced by recombinant DNAtechnique in bacterial host cells. The peptide is produced as a fusionprotein, chosen to assist in transporting the fusion peptide toinclusion bodies, periplasm, outer membrane or extracellularenvironment.

Construction of Plasmids Encoding MBI-11 Peptide Fusion Protein

Amplification by polymerase chain reaction is used to synthesizedouble-stranded DNA encoding the MBI peptide genes from single-strandedtemplates. For MBI-11, 100 μl of reaction mix is prepared containing 50to 100 ng of template, 25 pmole of each primer, 1.5 mM MgCl₂, 200 μM ofeach dNTP, 2U of Taq polymerase in buffer supplied by the manufacturer.Amplification conditions are 25 cycles of 94° C. for 30 sec., 55° C. for30 sec., 74° C. for 30 sec., followed by 74° C. for 1 min. Amplifiedproduct is digested with BamHI and HindIII and cloned into a plasmidexpression vector encoding the fusion partner and a suitable selectionmarker.

Production of MBI-11 Peptide Fusion in E. coli

The plasmid pR2h-11, employing a T7 promoter, high copy origin ofreplication, Ap^(r) marker and containing the gene of the fusionprotein, is co-electroporated with pGP1-2 into E. coli strain XL1-Blue.Plasmid pGP1-2 contains a T7 RNA polymerase gene under control of alambda promoter and cl857 repressor gene. Fusion protein expression isinduced by a temperature shift from 30° C. to 42° C. Inclusion bodiesare washed with solution containing solubilizer and extracted withorganic extraction solvent. Profiles of the samples are analyzed bySDS-PAGE. FIG. 1 shows the SDS-PAGE analysis and an extraction profileof inclusion body from whole cell. The major contaminant in the organicsolvent extracted material is β-lactamase (FIG. 1). The expression levelin these cells is presented in Table 10.

TABLE 10 % which is Fusion Mol. mass % protein in whole % in inclusionMBI-11 protein (kDa) cell lysate body extract peptide MBI-11 20.1 15 427.2

In addition, a low-copy-number vector, pPD100, which has achloramphenicol resistance gene, is used to express MBI-11 in order toeliminate the need for using ampicillin, thereby reducing the appearanceof β-lactamase in extracted material. This plasmid allows selective geneexpression and high-level protein overproduction in E. coli using thebacteriophage T7 RNA polymerase/T7 promoter system (Dersch et al., FEMSMicrobiol. Lett. 123: 19-26, 1994). pPD100 contains a chloramphenicolresistance gene (CAT) as a selective marker, a multiple cloning site,and an on sequence derived from the low-copy-number vector pSC101. Thereare only about 4 to 6 copies of these plasmids per host cell. Theresulting construct containing MBI-11 is called pPDR2h-11. FIG. 2presents a gel electrophoresis analysis of the MBI-11 fusion proteinexpressed in this vector. Expression level of MBI-11 fusion protein iscomparable with that obtained from plasmid pR2h-11. The CAT gene productis not apparent, presumably due to the low-copy-number nature of thisplasmid, CAT protein is not expressed at high levels in pPDR2h-11.

Example 4 In Vitro Assays to Measure Cationic Peptide Activity

A cationic peptide may be tested for antimicrobial activity alone beforeassessing its enhancing activity with antibiotic agents. Preferably, thepeptide has measurable antimicrobial activity.

Agarose Dilution Assay

The agarose dilution assay measures antimicrobial activity of peptidesand peptide analogues, which is expressed as the minimum inhibitoryconcentration (MIC) of the peptides.

In order to mimic in vivo conditions, calcium and magnesium supplementedMueller Hinton broth is used in combination with a low EEO agarose asthe bacterial growth medium. Agarose, rather than agar, is used as thecharged groups in agar prevent peptide diffusion through the media. Themedia is autoclaved and then cooled to 50-55° C. in a water bath beforeaseptic addition of antimicrobial solutions. The same volume ofdifferent concentrations of peptide solution are added to the cooledmolten agarose that is then poured to a depth of 3-4 mm.

The bacterial inoculum is adjusted to a 0.5 McFarland turbidity standard(PML Microbiological) and then diluted 1:10 before application on to theagarose plate. The final inoculum applied to the agarose isapproximately 10⁴ CFU in a 5-8 mm diameter spot. The agarose plates areincubated at 35-37° C. for 16 to 20 hours.

The MIC is recorded as the lowest concentration of peptide thatcompletely inhibits growth of the organism as determined by visualinspection. Representative MICs for various indolicidin analoguesagainst bacteria are shown in Table 11 and representative MICs againstCandida are shown in Table 12 below.

TABLE 11 Organism Organism # MIC (μg/ml) 1. MBI 10 A. calcoaceticusAC001 128 E. coli ECO002 128 E. faecalis EFS004 8 K. pneumoniae KP001128 P. aeruginosa PA003 >128 S. aureus SA007 2 S. maltophilia SMA001 128S. marcescens SMS003 >128 2. MBI 10A E. faecalis EFS004 16 E. faeciumEFM003 8 S. aureus SA010 8 3. MBI 10CN A. calcoaceticus AC001 64 E.cloacae ECL007 >128 E. coli ECO001 32 E. coli SBECO2 16 E. faecalisEFS004 8 E. faecium EFM003 2 K. pneumoniae KP002 64 P. aeruginosaPA002 >128 S. aureus SA003 2 S. epidermidis SE010 4 S. maltophiliaSMA002 64 S. marcescens SMS004 >128 4. MBI 11 A. calcoaceticus AC002 8E. cloacae ECL007 >128 E. coli ECO002 64 E. faecium EFM003 4 E. faecalisEFS002 64 K. pneumoniae KP001 128 P. aeruginosa PA004 >128 S. aureusSA004 4 S. maltophilia SMA002 128 S. marcescens SMS004 >128 5. MBI 11AA. calcoaceticus AC001 >64 E. cloacae ECL007 >64 E. coli ECO005 >64 E.faecalis EFS004 32 K. pneumoniae KP001 64 P. aeruginosa PA024 >64 S.aureus SA002 4 S. maltophilia SMA002 >64 S. marcescens SMS003 >64 6. MBI11ACN A. calcoaceticus AC002 2 E. cloacae ECL007 >128 E. coli ECO005 16E. faecalis EFS004 8 E. faecalis EFS008 64 K. pneumoniae KP001 16 P.aeruginosa PA004 >128 S. aureus SA014 8 S. epidermidis SE010 4 S.maltophilia SMA002 64 S. marcescens SMS003 >128 7. MBI 11CN A.calcoaceticus AC001 128 E. cloacae ECL007 >64 E. coli ECO002 8 E.faecium EFM001 8 E. faecalis EFS001 32 H. influenzae HIN001 >128 K.pneumoniae KP002 128 P. aeruginosa PA003 >128 P. mirabilis PM002 >128 S.aureus SA003 2 S. marcescens SBSM1 >128 S. pneumoniae SBSPN2 >128 S.epidermidis SE001 2 S. maltophilia SMA001 64 S. marcescens SMS003 >128S. pyogenes SPY003 8 8. MBI 11CNR A. calcoaceticus AC002 4 E. cloacaeECL007 >128 E. coli ECO005 8 E. faecalis EFS001 4 K. pneumoniae KP001 4P. aeruginosa PA004 32 S. aureus SA093 4 S. epidermidis SE010 4 S.maltophilia SMA002 32 S. marcescens SMS003 128 9. MBI 11CNW1 A.calcoaceticus AC002 8 E. cloacae ECL007 64 E. coli ECO005 32 E. faecalisEFS001 8 K. pneumoniae KP001 32 P. aeruginosa PA004 64 S. aureus SA010 4S. maltophilia SMA002 32 S. marcescens SMS003 >128 10. MBI 11CNX1 A.calcoaceticus AC001 >64 E. cloacae ECL007 >64 E. coli ECO005 64 E.faecalis EFS004 16 K. pneumoniae KP001 >64 P. aeruginosa PA024 >64 S.aureus SA006 2 S. maltophilia SMA002 >64 S. marcescens SMS003 >64 11.MBI 11CNY1 A. calcoaceticus AC001 >64 E. cloacae ECL007 >64 E. coliECO005 >64 E. faecalis EFS004 >64 K. pneumoniae KP001 >64 P. aeruginosaPA004 >64 S. aureus SA006 16 S. epidermidis SE010 128 S. maltophiliaSMA002 >64 S. marcescens SMS003 >64 12. MBI 11M4 E. faecium EFM001 32 E.faecalis EFS001 32 S. aureus SA008 8 13. MBI 11M8 E. faecalis EFS002 32E. faecium EFM002 32 S. aureus SA008 32 14. MBI 11A1CN A. calcoaceticusAC002 16 E. cloacae ECL007 >128 E. coli ECO002 32 E. faecium EFM002 1 E.faecalis EFS002 32 H. influenzae HIN002 >128 K. pneumoniae KP002 >128 P.aeruginosa PA004 >128 S. aureus SA005 8 P. vulgaris SBPV1 >128 S.marcescens SBSM2 >128 S. pneumoniae SBSPN2 >128 S. epidermidis SE002 16S. maltophilia SMA002 >128 15. MBI 11A2CN A. calcoaceticus AC001 >128 E.cloacae ECL007 >128 E. coli ECO003 >128 E. faecium EFM003 16 E. faecalisEFS002 >128 K. pneumoniae KP002 >128 P. aeruginosa PA004 >128 S. aureusSA004 8 S. maltophilia SMA001 >128 S. marcescens SMS003 >128 16. MBI11A3CN A. calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coliECO002 >128 E. faecium EFM003 64 E. faecalis EFS002 >128 H. influenzaeHIN002 >128 K. pneumoniae KP001 >128 P. aeruginosa PA002 >128 S. aureusSA004 32 P. vulgaris SBPV1 >128 S. marcescens SBSM2 >128 S. pneumoniaeSBSPN3 >128 S. epidermidis SE002 128 S. maltophilia SMA001 >128 17. MBI11A4CN A. calcoaceticus AC002 8 E. cloacae ECL007 >128 E. coli ECO003 32E. faecalis EFS002 64 E. faecium EFM001 32 K. pneumoniae KP001 >128 P.aeruginosa PA004 >128 S. aureus SA005 2 S. epidermidis SE002 8 S.maltophilia SMA002 >128 S. marcescens SMS004 >128 18. MBI 11A5CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli ECO003 128 E.faecium EFM003 4 E. faecalis EFS002 32 K. pneumoniae KP001 >128 P.aeruginosa PA003 >128 S. aureus SA002 16 S. maltophilia SMA002 >128 S.marcescens SMS003 >128 19. MBI 11A6CN E. faecium EFM003 2 E. faecalisEFS004 64 S. aureus SA016 2 20. MBI 11A7CN E. faecium EFM003 2 E.faecalis EFS002 16 S. aureus SA009 2 21. MBI 11A8CN A. calcoaceticusAC002 8 E. cloacae ECL007 >128 E. coli ECO005 32 E. faecalis EFS001 4 K.pneumoniae KP001 128 P. aeruginosa PA004 >128 S. aureus SA093 1 S.epidermidis SE010 16 S. maltophilia SMA002 32 S. marcescens SMS003 >12822. MBI 11B1CN A. calcoaceticus AC001 32 E. cloacae ECL007 >128 E. coliECO003 8 E. faecium EFM002 2 E. faecalis EFS004 8 K. pneumoniae KP002 64P. aeruginosa PA005 >128 S. aureus SA005 2 S. epidermidis SE001 2 S.maltophilia SMA001 64 S. marcescens SMS004 >128 23. MBI 11B1CNW1 A.calcoaceticus AC002 16 E. cloacae ECL007 64 E. coli ECO005 32 E.faecalis EFS004 8 K. pneumoniae KP001 32 P. aeruginosa PA004 64 S.aureus SA014 16 S. epidermidis SE010 8 S. maltophilia SMA002 32 S.marcescens SMS003 >128 24. MBI 11B2CN A. calcoaceticus AC001 64 E.cloacae ECL007 >128 E. coli ECO003 16 E. faecium EFM001 8 E. faecalisEFS004 8 K. pneumoniae KP002 64 P. aeruginosa PA003 >128 S. aureus SA0052 S. maltophilia SMA002 64 S. marcescens SMS004 >128 25. MBI 11B3CN A.calcoaceticus AC001 64 E. cloacae ECL007 >128 E. coli ECO002 16 E.faecium EFM001 8 E. faecalis EFS001 16 K. pneumoniae KP002 64 P.aeruginosa PA003 >128 S. aureus SA010 4 S. maltophilia SMA002 32 S.marcescens SMS004 >128 26. MBI 11B4CN A. calcoaceticus AC001 >128 E.cloacae ECL007 >128 E. coli ECO003 16 E. faecalis EFS002 16 H.influenzae HIN002 >128 K. pneumoniae KP002 128 P. aeruginosa PA006 >128S. aureus SA004 2 S. marcescens SBSM2 >128 S. pneumoniae SBSPN3 128 S.epidermidis SE010 4 S. maltophilia SMA002 64 S. marcescens SMS004 >12827. MBI 11B4ACN A. calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coliECO005 32 E. faecalis EFS008 64 K. pneumoniae KP001 32 P. aeruginosaPA004 >128 S. aureus SA008 1 S. epidermidis SE010 8 S. maltophiliaSMA002 64 S. marcescens SMS003 >128 28. MBI 11B5CN E. faecium EFM002 1E. faecalis EFS002 16 S. aureus SA005 2 29. MBI 11B7 A. calcoaceticusAC002 4 E. cloacae ECL007 >128 E. coli ECO005 16 E. faecalis EFS008 8 K.pneumoniae KP001 16 P. aeruginosa PA004 >128 S. aureus SA093 1 S.epidermidis SE010 4 S. maltophilia SMA002 64 S. marcescens SMS003 >12830. MBI 11B7CN A. calcoaceticus AC003 32 E. cloacae ECL009 32 E. coliECO002 8 E. faecium EFM001 4 E. faecalis EFS004 4 H. influenzaeHIN002 >128 K. pneumoniae KP001 32 P. aeruginosa PA004 128 P. mirabilisPM002 >128 S. aureus SA009 2 S. marcescens SBSM1 >128 S. pneumoniaeSBSPN3 >128 S. epidermidis SE003 2 S. maltophilia SMA004 128 S. pyogenesSPY006 16 31. MBI 11B7CNR A. calcoaceticus AC002 4 E. cloacae ECL007 64E. coli ECO005 8 E. faecalis EFS001 4 K. pneumoniae KP001 8 P.aeruginosa PA004 64 S. aureus SA093 2 S. epidermidis SE010 4 S.maltophilia SMA002 32 S. marcescens SMS003 >128 32. MBI 11B8CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli ECO002 16 E.faecium EFM001 16 E. faecalis EFS002 32 K. pneumoniae KP001 >128 P.aeruginosa PA005 >128 S. aureus SA009 4 S. epidermidis SE002 4 S.maltophilia SMA002 128 S. marcescens SMS003 >128 33. MBI 11B9CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO005 8 E. faeciumEFM002 4 E. faecalis EFS002 8 H. influenzae HIN002 >128 K. pneumoniaeKP001 32 P. aeruginosa PA004 128 P. mirabilis PM002 >128 S. aureus SA0104 S. pneumoniae SBSPN2 >128 S. epidermidis SE010 2 S. maltophilia SMA00232 S. marcescens SMS003 >128 S. pneumoniae SPN044 >128 S. pyogenesSPY005 16 34. MBI 11B9ACN A. calcoaceticus AC001 32 E. cloacaeECL007 >128 E. coli ECO003 8 E. faecium EFM001 4 E. faecalis EFS004 8 K.pneumoniae KP002 32 P. aeruginosa PA005 >128 S. aureus SA019 2 S.epidermidis SE002 2 S. maltophilia SMA001 16 S. marcescens SMS004 >12835. MBI 11B10CN E. faecium EFM003 4 E. faecalis EFS002 64 S. aureusSA008 2 36. MBI 11B16CN A. calcoaceticus AC002 4 E. cloacae ECL007 >128E. coli ECO005 16 E. faecalis EFS001 2 K. pneumoniae KP001 16 P.aeruginosa PA004 >128 S. aureus SA093 2 S. epidermidis SE010 4 S.maltophilia SMA002 32 S. marcescens SMS003 >128 37. MBI 11B17CN A.calcoaceticus AC002 2 E. cloacae ECL007 >128 E. coli ECO005 8 E.faecalis EFS008 4 K. pneumoniae KP001 16 P. aeruginosa PA004 >128 S.aureus SA093 2 S. epidermidis SE010 4 S. maltophilia SMA002 32 S.marcescens SMS003 >128 38. MBI 11B18CN A. calcoaceticus AC002 2 E.cloacae ECL007 >128 E. coli ECO005 32 E. faecalis EFS008 4 K. pneumoniaeKP001 32 P. aeruginosa PA004 >128 S. aureus SA093 2 S. epidermidis SE0104 S. maltophilia SMA002 64 S. marcescens SMS003 >128 39. MBI 11C3CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO002 16 E.faecium EFM002 1 E. faecalis EFS002 32 K. pneumoniae KP001 128 P.aeruginosa PA005 >128 S. aureus SA005 2 S. epidermidis SE002 2 S.maltophilia SMA002 64 S. marcescens SMS004 >128 40. MBI 11C4CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO005 32 E.faecium EFM003 2 E. faecalis EFS002 32 K. pneumoniae KP001 >128 P.aeruginosa PA005 >128 S. aureus SA009 4 S. epidermidis SE002 4 S.maltophilia SMA002 64 S. marcescens SMS004 >128 41. MBI 11C5CN A.calcoaceticus AC001 32 E. cloacae ECL007 >128 E. coli ECO001 8 E.faecium EFM003 2 E. faecalis EFS002 16 K. pneumoniae KP002 16 P.aeruginosa PA003 64 S. aureus SA009 2 S. epidermidis SE002 2 S.maltophilia SMA002 16 S. marcescens SMS004 >128 42. MBI 11D1CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli ECO002 16 E.faecium EFM001 16 E. faecalis EFS002 32 K. pneumoniae KP002 64 P.aeruginosa PA003 >128 S. aureus SA004 2 S. epidermidis SE010 8 S.maltophilia SMA001 64 S. marcescens SMS003 >128 43. MBI 11D3CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli ECO002 64 E.faecium EFM003 8 E. faecalis EFS002 32 K. pneumoniae KP002 >128 P.aeruginosa PA024 >128 S. aureus SA009 8 S. maltophilia SMA001 64 S.marcescens SMS004 >128 44. MBI 11D4CN A. calcoaceticus AC001 >64 E.cloacae ECL007 >64 E. coli ECO003 64 E. faecium EFM002 1 E. faecalisEFS002 16 K. pneumoniae KP002 >64 P. aeruginosa PA004 >64 S. aureusSA009 4 S. maltophilia SMA001 >64 S. marcescens SMS004 >64 45. MBI11D5CN A. calcoaceticus AC001 >64 E. cloacae ECL007 >64 E. coli ECO00364 E. faecium EFM003 1 E. faecalis EFS002 16 K. pneumoniae KP001 >64 P.aeruginosa PA003 >64 S. aureus SA005 8 S. maltophilia SMA001 64 S.marcescens SMS004 >64 46. MBI 11D6CN A. calcoaceticus AC002 4 E. cloacaeECL007 >32 E. coli ECO002 32 E. faecium EFM003 1 E. faecalis EFS002 4 K.pneumoniae KP002 >64 P. aeruginosa PA024 >64 S. aureus SA009 8 S.epidermidis SE010 4 S. maltophilia SMA001 >64 S. marcescens SMS004 >6447. MBI 11D9M8 E. faecium EFM002 32 S. aureus SA007 32 E. faecalisEFS002 128 S. aureus SA016 128 48. MBI 11D10M8 E. faecium EFM003 32 E.faecalis EFS002 32 S. aureus SA008 32 49. MBI 11D11H A. calcoaceticusAC001 >64 E. cloacae ECL007 >64 E. coli ECO002 32 K. pneumoniaeKP001 >64 P. aeruginosa PA001 >64 S. aureus SA008 4 S. maltophiliaSMA002 >64 S. marcescens SMS004 >64 50. MBI 11D12H A. calcoaceticusAC001 >64 E. cloacae ECL007 >64 E. coli ECO003 64 E. faecalis EFS004 16K. pneumoniae KP002 >64 P. aeruginosa PA004 >64 S. aureus SA014 16 S.maltophilia SMA002 >64 S. marcescens SMS004 >64 51. MBI 11D13H A.calcoaceticus AC001 64 E. cloacae ECL007 >64 E. coli ECO002 32 E.faecalis EFS004 16 K. pneumoniae KP002 >64 P. aeruginosa PA004 >64 S.aureus SA025 4 S. maltophilia SMA002 >64 S. marcescens SMS004 >64 52.MBI 11D14CN E. faecium EFM003 1 E. faecalis EFS002 32 S. aureus SA009 453. MBI 11D15CN E. faecium EFM003 4 E. faecalis EFS002 32 S. aureusSA009 8 54. MBI 11D18CN A. calcoaceticus AC003 32 E. cloacae ECL009 64E. coli ECO002 4 E. faecium EFM003 2 E. faecalis EFS002 32 H. influenzaeHIN002 >128 K. pneumoniae KP002 64 P. aeruginosa PA006 >128 P. mirabilisPM003 >128 S. aureus SA010 4 P. vulgaris SBPV1 32 S. marcescensSBSM2 >128 S. pneumoniae SBSPN3 64 S. epidermidis SE010 2 S. maltophiliaSMA003 16 S. pyogenes SPY003 32 55. MBI 11E1CN A. calcoaceticus AC001 32E. cloacae ECL007 >128 E. coli ECO003 8 E. faecium EFM001 8 E. faecalisEFS002 8 K. pneumoniae KP002 32 P. aeruginosa PA003 128 S. aureus SA0061 S. maltophilia SMA001 64 S. marcescens SMS003 >128 56. MBI 11E2CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO002 8 E. faeciumEFM001 16 E. faecalis EFS002 32 K. pneumoniae KP002 64 P. aeruginosaPA001 >128 S. aureus SA016 2 S. epidermidis SE010 4 S. maltophiliaSMA001 64 S. marcescens SMS004 >128 57. MBI 11E3CN A. calcoaceticusAC001 16 E. cloacae ECL007 >128 E. coli ECO001 4 E. faecium EFM003 2 E.faecalis EFS004 8 H. influenzae HIN002 >128 K. pneumoniae KP002 32 P.aeruginosa PA041 64 P. mirabilis PM001 >128 S. aureus SA010 2 S.pneumoniae SBSPN2 >128 S. epidermidis SE002 1 S. maltophilia SMA001 32S. marcescens SMS004 >128 S. pneumoniae SPN044 >128 S. pyogenes SPY00216 58. MBI 11F1CN E. cloacae ECL007 >128 E. coli ECO003 8 E. faeciumEFM003 2 E. faecalis EFS004 16 K. pneumoniae KP002 32 P. aeruginosaPA004 64 S. aureus SA009 2 S. marcescens SBSM1 >128 S. marcescensSMS003 >128 59. MBI 11F2CN A. calcoaceticus AC002 4 E. coli ECO002 8 E.faecium EFM002 4 E. faecalis EFS002 32 K. pneumoniae KP002 128 P.aeruginosa PA005 >128 S. aureus SA012 4 S. epidermidis SE002 4 S.maltophilia SMA002 64 S. marcescens SMS004 >128 60. MBI 11F3CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO002 8 E. faeciumEFM003 4 E. faecalis EFS002 8 H. influenzae HIN002 >128 K. pneumoniaeKP002 64 P. aeruginosa PA041 128 S. aureus SA005 2 S. pneumoniaeSBSPN3 >128 S. epidermidis SE003 2 S. maltophilia SMA002 64 S.marcescens SMS004 >128 S. pneumoniae SPN044 >128 S. pyogenes SPY006 861. MBI 11F4CN A. calcoaceticus AC003 16 E. cloacae ECL006 16 E. coliECO001 8 E. faecalis EFS004 8 H. influenzae HIN003 >128 K. pneumoniaeKP001 8 P. aeruginosa PA020 32 S. aureus SA007 1 S. marcescensSBSM1 >128 S. pneumoniae SBSPN3 >128 S. epidermidis SE010 2 S.maltophilia SMA006 16 S. pyogenes SPY005 32 62. MBI 11F4CNR A.calcoaceticus AC002 16 E. cloacae ECL007 32 E. coli ECO005 32 E.faecalis EFS008 32 K. pneumoniae KP001 32 P. aeruginosa PA004 64 S.aureus SA093 8 S. epidermidis SE010 8 S. maltophilia SMA002 32 S.marcescens SMS003 >128 63. MBI 11G2CN E. cloacae ECL007 >128 E. coliECO003 16 E. faecium EFM002 4 E. faecalis EFS004 16 K. pneumoniae KP002128 P. aeruginosa PA004 >128 S. aureus SA009 2 S. maltophiliaSMA001 >128 S. marcescens SMS004 >128 64. MBI 11G3CN E. cloacaeECL007 >128 E. coli ECO003 64 E. faecium EFM002 32 E. faecalis EFS002 64K. pneumoniae KP001 >128 P. aeruginosa PA003 >128 S. aureus SA009 8 S.maltophilia SMA001 >128 S. marcescens SMS004 >128 65. MBI 11G4CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO005 32 E.faecium EFM003 1 E. faecalis EFS002 32 K. pneumoniae KP001 >128 P.aeruginosa PA004 >128 S. aureus SA004 1 S. epidermidis SE010 2 S.maltophilia SMA002 64 S. marcescens SMS003 >128 66. MBI 11G5CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO003 16 E.faecium EFM002 8 E. faecalis EFS002 16 K. pneumoniae KP001 >128 P.aeruginosa PA003 >128 S. aureus SA012 4 S. epidermidis SE002 2 S.maltophilia SMA002 64 S. marcescens SMS004 >128 67. MBI 11G6CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli ECO002 32 E.faecium EFM003 4 E. faecalis EFS002 128 K. pneumoniae KP001 >128 P.aeruginosa PA004 >128 S. aureus SA006 2 S. epidermidis SE002 8 S.maltophilia SMA001 >128 S. marcescens SMS003 >128 68. MBI 11G6ACN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO005 64 E.faecalis EFS008 >128 K. pneumoniae KP001 >128 P. aeruginosa PA004 >128S. aureus SA014 64 S. epidermidis SE010 32 S. maltophilia SMA002 >128 S.marcescens SMS003 >128 69. MBI 11G7CN A. calcoaceticus AC001 128 E.cloacae ECL006 64 E. coli ECO005 8 E. faecium EFM001 8 E. faecalisEFS002 32 H. influenzae HIN002 >128 K. pneumoniae KP001 16 P. aeruginosaPA006 >128 S. aureus SA012 2 H. influenzae SBHIN2 >128 S. marcescensSBSM1 >128 S. pneumoniae SBSPN2 >128 S. epidermidis SE002 2 S.maltophilia SMA001 32 S. marcescens SMS003 >128 S. pneumoniaeSPN044 >128 S. pyogenes SPY006 16 70. MBI 11G7ACN A. calcoaceticus AC0024 E. cloacae ECL007 >32 E. coli ECO002 16 E. faecium EFM001 8 E.faecalis EFS008 32 K. pneumoniae KP002 >32 P. aeruginosa PA006 >32 S.aureus SA010 1 S. epidermidis SE002 4 S. maltophilia SMA001 32 S.marcescens SMS004 >32 71. MBI 11G13CN E. coli ECO002 32 E. faeciumEFM002 16 E. faecalis EFS002 64 H. influenzae HIN002 >128 P. aeruginosaPA004 >128 S. aureus SA004 4 E. coli SBECO3 32 S. marcescens SBSM1 >128S. pneumoniae SBSPN3 128 72. MBI 11G14CN A. calcoaceticus AC002 8 E.cloacae ECL007 >128 E. coli ECO003 32 E. faecium EFM001 16 E. faecalisEFS002 32 K. pneumoniae KP002 128 P. aeruginosa PA006 >128 S. aureusSA013 0.5 S. epidermidis SE002 8 S. maltophilia SMA002 128 S. marcescensSMS004 >128 73. MBI 11G16CN A. calcoaceticus AC002 8 E. cloacaeECL007 >128 E. coli ECO005 16 E. faecalis EFS008 16 K. pneumoniae KP00116 P. aeruginosa PA004 128 S. aureus SA093 2 S. epidermidis SE010 4 S.maltophilia SMA002 64 S. marcescens SMS003 >128 MIC (□g/ml) 74. MBI11A6CN A. calcoaceticus AC002 2 E. cloacae ECL007 >128 E. coli ECO005 16E. faecalis EFS001 1 E. faecalis EFS008 8 K. pneumoniae KP001 32 P.aeruginosa PA004 >128 S. aureus SA014 4 S. aureus SA093 0.5 S.epidermidis SE010 4 S. maltophilia SMA002 128 S. marcescens SMS003 >12875. MBI 11A7CN A. calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coliECO005 8 E. faecalis EFS001 2 E. faecalis EFS008 4 K. pneumoniae KP001 8P. aeruginosa PA004 >128 S. aureus SA014 4 S. aureus SA093 1 S.epidermidis SE010 4 S. maltophilia SMA002 64 S. marcescens SMS003 >12876. MBI 11A9CN A. calcoaceticus AC002 8 E. cloacae ECL007 128 E. coliECO005 32 E. faecalis EFS001 2 E. faecalis EFS008 8 K. pneumoniae KP00132 P. aeruginosa PA004 128 S. aureus SA014 4 S. aureus SA093 2 S.epidermidis SE010 4 S. maltophilia SMA002 32 S. marcescens SMS003 >12877. MBI 11A10CN A. calcoaceticus AC002 4 E. cloacae ECL007 64 E. coliECO005 16 E. faecalis EFS001 4 E. faecalis EFS008 16 K. pneumoniae KP00116 P. aeruginosa PA004 64 S. aureus SA014 4 S. aureus SA093 2 S.epidermidis SE010 4 S. maltophilia SMA002 32 S. marcescens SMS003 >12878. MBI 11B5CN A. calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coliECO005 16 E. faecalis EFS001 2 E. faecalis EFS008 8 K. pneumoniae KP00116 P. aeruginosa PA004 >128 S. aureus SA014 2 S. aureus SA093 1 S.epidermidis SE010 1 S. maltophilia SMA002 64 S. marcescens SMS003 >12879. MBI 11B7ACN A. calcoaceticus AC002 2 E. cloacae ECL007 >128 E. coliECO005 8 E. faecalis EFS001 1 E. faecalis EFS008 8 K. pneumoniae KP001 8P. aeruginosa PA004 128 S. aureus SA014 2 S. aureus SA093 1 S.epidermidis SE010 4 S. maltophilia SMA002 32 S. marcescens SMS003 >12880. MBI 11B7CNF12 A. calcoaceticus AC002 4 E. cloacae ECL007 >128 E.coli ECO005 16 E. faecalis EFS001 2 E. faecalis EFS008 8 K. pneumoniaeKP001 16 P. aeruginosa PA004 >128 S. aureus SA014 2 S. aureus SA093 1 S.epidermidis SE010 4 S. maltophilia SMA002 64 S. marcescens SMS003 >12881. MBI 11B10CN A. calcoaceticus AC002 2 E. cloacae ECL007 >128 E. coliECO005 16 E. faecalis EFS001 8 E. faecalis EFS008 64 K. pneumoniae KP00132 P. aeruginosa PA004 >128 S. aureus SA014 4 S. aureus SA093 1 S.epidermidis SE010 4 S. maltophilia SMA002 128 S. marcescens SMS003 >12882. MBI 11B19CN A. calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coliECO005 8 E. faecalis EFS001 2 E. faecalis EFS008 32 K. pneumoniae KP00164 P. aeruginosa PA004 128 S. aureus SA014 4 S. aureus SA093 2 S.epidermidis SE010 4 S. maltophilia SMA002 32 S. marcescens SMS003 >12883. MBI 11B20 A. calcoaceticus AC002 32 E. cloacae ECL007 128 E. coliECO005 32 E. faecalis EFS001 8 E. faecalis EFS008 32 K. pneumoniae KP00164 P. aeruginosa PA004 128 S. aureus SA014 32 S. aureus SA093 4 S.epidermidis SE010 32 S. maltophilia SMA002 64 S. marcescens SMS003 >12884. MBI 11D9M8 A. calcoaceticus AC002 128 E. cloacae ECL007 >128 E. coliECO005 >128 E. faecalis EFS001 8 E. faecalis EFS008 128 K. pneumoniaeKP001 >128 P. aeruginosa PA004 >128 S. aureus SA014 128 S. aureus SA0938 S. epidermidis SE010 128 S. maltophilia SMA002 >128 S. marcescensSMS003 >128 85. MBI 11D19CN A. calcoaceticus AC002 8 E. cloacaeECL007 >128 E. coli ECO005 32 E. faecalis EFS001 4 E. faecalis EFS008 64K. pneumoniae KP001 32 P. aeruginosa PA004 128 S. aureus SA014 4 S.aureus SA093 2 S. epidermidis SE010 8 S. maltophilia SMA002 64 S.marcescens SMS003 >128 86. MBI 11F4 A. calcoaceticus AC002 4 E. cloacaeECL007 128 E. coli ECO005 8 E. faecalis EFS001 2 E. faecalis EFS008 8 K.pneumoniae KP001 8 P. aeruginosa PA004 128 S. aureus SA014 2 S. aureusSA093 1 S. epidermidis SE010 4 S. maltophilia SMA002 16 S. marcescensSMS003 >128 87. MBI 11F5CN A. calcoaceticus AC002 4 E. cloacae ECL007128 E. coli ECO005 8 E. faecalis EFS001 2 E. faecalis EFS008 8 K.pneumoniae KP001 8 P. aeruginosa PA004 32 S. aureus SA014 4 S. aureusSA093 2 S. epidermidis SE010 4 S. maltophilia SMA002 16 S. marcescensSMS003 >128 88. MBI 11F6CN A. calcoaceticus AC002 16 E. cloacae ECL00764 E. coli ECO005 32 E. faecalis EFS001 16 E. faecalis EFS008 16 K.pneumoniae KP001 32 P. aeruginosa PA004 128 S. aureus SA014 16 S. aureusSA093 8 S. epidermidis SE010 8 S. maltophilia SMA002 64 S. marcescensSMS003 >128 89. MBI 11G24CN A. calcoaceticus AC002 4 E. cloacaeECL007 >128 E. coli ECO005 16 E. faecalis EFS001 2 E. faecalis EFS008 8K. pneumoniae KP001 16 P. aeruginosa PA004 128 S. aureus SA014 2 S.aureus SA093 1 S. epidermidis SE010 4 S. maltophilia SMA002 32 S.marcescens SMS003 >128 90. MBI 11G25CN A. calcoaceticus AC002 2 E.cloacae ECL007 >128 E. coli ECO005 8 E. faecalis EFS001 2 E. faecalisEFS008 16 K. pneumoniae KP001 16 P. aeruginosa PA004 64 S. aureus SA0142 S. aureus SA093 1 S. epidermidis SE010 2 S. maltophilia SMA002 32 S.marcescens SMS003 >128 91. MBI 11G26CN A. calcoaceticus AC002 2 E.cloacae ECL007 >128 E. coli ECO005 32 E. faecalis EFS001 2 E. faecalisEFS008 4 K. pneumoniae KP001 32 P. aeruginosa PA004 >128 S. aureus SA0144 S. aureus SA093 0.5 S. epidermidis SE010 4 S. maltophilia SMA002 128S. marcescens SMS003 >128 92. MBI 11G27CN A. calcoaceticus AC002 2 E.cloacae ECL007 >128 E. coli ECO005 16 E. faecalis EFS001 8 E. faecalisEFS008 32 K. pneumoniae KP001 32 P. aeruginosa PA004 128 S. aureus SA0144 S. aureus SA093 1 S. epidermidis SE010 8 S. maltophilia SMA002 32 S.marcescens SMS003 >128 93. MBI 11G28CN A. calcoaceticus AC002 2 E.cloacae ECL007 >128 E. coli ECO005 8 E. faecalis EFS001 4 E. faecalisEFS008 32 K. pneumoniae KP001 64 P. aeruginosa PA004 128 S. aureus SA0144 S. aureus SA093 1 S. epidermidis SE010 4 S. maltophilia SMA002 32 S.marcescens SMS003 >128 94. MBI 11H01CN A. calcoaceticus AC002 2 E.cloacae ECL007 >128 E. coli ECO005 8 E. faecalis EFS001 2 E. faecalisEFS008 8 K. pneumoniae KP001 8 P. aeruginosa PA004 128 S. aureus SA014 2S. aureus SA093 1 S. epidermidis SE010 2 S. maltophilia SMA002 32 S.marcescens SMS003 >128 95. MBI 11H02CN A. calcoaceticus AC002 4 E.cloacae ECL007 >128 E. coli ECO005 16 E. faecalis EFS001 2 E. faecalisEFS008 16 K. pneumoniae KP001 32 P. aeruginosa PA004 >128 S. aureusSA014 2 S. aureus SA093 1 S. epidermidis SE010 4 S. maltophilia SMA00264 S. marcescens SMS003 >128 96. MBI 11H03CN A. calcoaceticus AC002 8 E.cloacae ECL007 >128 E. coli ECO005 16 E. faecalis EFS001 2 E. faecalisEFS008 8 K. pneumoniae KP001 16 P. aeruginosa PA004 >128 S. aureus SA0142 S. aureus SA093 2 S. epidermidis SE010 4 S. maltophilia SMA002 64 S.marcescens SMS003 >128 97. MBI 11H04CN A. calcoaceticus AC002 8 E.cloacae ECL007 >128 E. coli ECO005 32 E. faecalis EFS001 2 E. faecalisEFS008 8 K. pneumoniae KP001 64 P. aeruginosa PA004 >128 S. aureus SA0144 S. aureus SA093 2 S. epidermidis SE010 16 S. maltophilia SMA002 >128S. marcescens SMS003 >128 98. MBI 11H05CN A. calcoaceticus AC002 4 E.cloacae ECL007 >128 E. coli ECO005 8 E. faecalis EFS001 2 E. faecalisEFS008 8 K. pneumoniae KP001 8 P. aeruginosa PA004 128 S. aureus SA014 2S. aureus SA093 1 S. epidermidis SE010 4 S. maltophilia SMA002 16 S.marcescens SMS003 >128 99. MBI 11H06CN A. calcoaceticus AC002 8 E.cloacae ECL007 >128 E. coli ECO005 16 E. faecalis EFS001 2 E. faecalisEFS008 16 K. pneumoniae KP001 64 P. aeruginosa PA004 >128 S. aureusSA014 8 S. aureus SA093 1 S. epidermidis SE010 8 S. maltophilia SMA00264 S. marcescens SMS003 >128 100. MBI 11H07CN A. calcoaceticus AC002 8E. cloacae ECL007 >128 E. coli ECO005 32 E. faecalis EFS001 4 E.faecalis EFS008 16 K. pneumoniae KP001 128 P. aeruginosa PA004 >128 S.aureus SA014 8 S. aureus SA093 2 S. epidermidis SE010 16 S. maltophiliaSMA002 128 S. marcescens SMS003 >128 101. MBI 11H08CN A. calcoaceticusAC002 4 E. cloacae ECL007 >128 E. coli ECO005 8 E. faecalis EFS001 2 E.faecalis EFS008 8 K. pneumoniae KP001 32 P. aeruginosa PA004 >128 S.aureus SA014 4 S. aureus SA093 1 S. epidermidis SE010 4 S. maltophiliaSMA002 32 S. marcescens SMS003 >128 102. MBI 11H09CN A. calcoaceticusAC002 4 E. cloacae ECL007 >128 E. coli ECO005 32 E. faecalis EFS001 4 E.faecalis EFS008 64 K. pneumoniae KP001 64 P. aeruginosa PA004 >128 S.aureus SA014 8 S. aureus SA093 2 S. epidermidis SE010 16 S. maltophiliaSMA002 64 S. marcescens SMS003 >128 103. MBI 11H10CN A. calcoaceticusAC002 4 E. cloacae ECL007 >128 E. coli ECO005 16 E. faecalis EFS001 2 E.faecalis EFS008 8 K. pneumoniae KP001 16 P. aeruginosa PA004 >128 S.aureus SA014 4 S. aureus SA093 1 S. epidermidis SE010 4 S. maltophiliaSMA002 64 S. marcescens SMS003 >128 104. MBI 11H11CN A. calcoaceticusAC002 4 E. cloacae ECL007 >128 E. coli ECO005 16 E. faecalis EFS001 2 E.faecalis EFS008 8 K. pneumoniae KP001 16 P. aeruginosa PA004 >128 S.aureus SA014 4 S. aureus SA093 1 S. epidermidis SE010 4 S. maltophiliaSMA002 64 S. marcescens SMS003 >128 105. MBI 11H12CN A. calcoaceticusAC002 4 E. cloacae ECL007 >128 E. coli ECO005 16 E. faecalis EFS001 2 E.faecalis EFS008 8 K. pneumoniae KP001 16 P. aeruginosa PA004 >128 S.aureus SA014 2 S. aureus SA093 1 S. epidermidis SE010 4 S. maltophiliaSMA002 64 S. marcescens SMS003 >128 106. MBI 11J01CN A. calcoaceticusAC002 4 E. cloacae ECL007 >128 E. coli ECO005 64 E. faecalis EFS001 128E. faecalis EFS008 >128 K. pneumoniae KP001 >128 P. aeruginosaPA004 >128 S. aureus SA014 16 S. aureus SA093 2 S. epidermidis SE010 32S. maltophilia SMA002 >128 S. marcescens SMS003 >128 107. MBI 11J02CN A.calcoaceticus AC002 4 E. cloacae ECL007 64 E. coli ECO005 4 E. faecalisEFS001 4 E. faecalis EFS008 16 K. pneumoniae KP001 4 P. aeruginosa PA00432 S. aureus SA014 2 S. aureus SA093 1 S. epidermidis SE010 2 S.maltophilia SMA002 8 S. marcescens SMS003 >128

TABLE 12 MBI 11CN MBI 11B7CN Organism MIC (μg/ml) MIC (μg/ml) C.albicans CA001 128 64 C. albicans CA002 64 32 C. albicans CA003 128 64C. albicans CA004 64 32 C. albicans CA005 128 32 C. albicans CA006 12864 C. albicans CA007 128 64 C. albicans CA008 64 32 C. albicans CA009 6432 C. albicans CA010 128 64 C. albicans CA011 64 64 C. albicans CA012128 64 C. albicans CA013 128 64 C. albicans CA014 64 32 C. albicansCA015 128 64 C. albicans CA016 128 64 C. albicans CA017 128 64 C.albicans CA018 128 64 C. albicans CA019 128 64 C. albicans CA020 128 32C. albicans CA021 128 32 C. albicans CA022 32 32 C. albicans CA023 12864 C. albicans CA024 16 8 C. glabrata CGL001 >128 128 C. glabrataCGL002 >128 128 C. glabrata CGL003 >128 128 C. glabrata CGL004 >128 128C. glabrata CGL005 >128 128 C. glabrata CGL009 >128 128 C. glabrataCGL010 >128 128 C. krusei CKR001 0.5 1 C. tropicalis CTR001 4 4 C.tropicalis CTR002 4 8 C. tropicalis CTR003 8 8 C. tropicalis CTR004 4 8C. tropicalis CTR005 4 4 C. tropicalis CTR006 16 8 C. tropicalis CTR00716 8 C. tropicalis CTR008 8 4 C. tropicalis CTR009 8 4

Broth Dilution Assay

Typically 100 μl of calcium and magnesium supplemented Mueller Hintonbroth is dispensed into each well of a 96-well microtitre plate and 100μl volumes of two-fold serial dilutions of the peptide are preparedacross the plate. One row of wells receives no peptide and is used as agrowth control. Each well is inoculated with approximately 5×10⁵ CFU ofbacteria and the plate is incubated at 35-37° C. for 16-20 hours. TheMIC is recorded at the lowest concentration of peptide that completelyinhibits growth of the organism as determined by visual inspection.

For example, MIC values in μg/ml are established by broth dilution assay(Table 13) or by agarose dilution assay (Table 14) for a series ofcationic peptides against various bacterial strains.

TABLE 13 Organism MBI 11CN MBI 11A1CN A. calcoaceticus 8191 256 >256 E.cloacae 13047 >128 >256 E. coli KL4 64 256 E. coli DH1 64 128 E. coliECO003 64 >256 E. coli 25922 128 512 E. faecalis 29212 64 >256 K.pneumoniae 13883 >128 >256 K. pneumoniae B44 64 >256 P. aeruginosa H650256 >256 P. aeruginosa H652 256 >256 P. aeruginosa 27853 >128 >256 P.aeruginosa 9503024 >256 >256 P. aeruginosa 8509041 256 >256 P.aeruginosa 9308077 128 >256 S. aureus 25923 32 512 S. aureus 2721764 >256 S. aureus 33593 64 >256 S. aureus 29213 32 512 S. aureus 880901432 >256 S. aureus 8809025 64 >256 S. aureus 8402093 32 >256 S.maltophilia 13637 128 256 S. epidermidis 14990 8 512 S. maltophilia H36164 256 S. marcescens 13880 >128 >256 S. marcescens B21 >256 >256

TABLE 14 Peptide MIC values in ug/ml MBI MBI MBI MBI MBI MBI MBI MBI MBIOrganism 10CN 11CN 11A1CN 11A3CN 11B4CN 11B8CN 11D18CN 11F1CN 11G13CN E.coli ATCC 25922 16 16 128 >128 32 32 16 8 64 E. coli ESS ND ND 16 >128 8ND 2 ND 32 E. coli NCTC 10418 8 4 16 64 8 4 2 2 16 E. faecium ATCC 292124 8 128 >128 8 8 8 8 32 P. aeruginosa ATCC >128 >128 >128 >128 >128 >128128 64 >128 27853 P. aeruginosa NCTC >128 >128 >128 >128 >128 >128 12864 >128 10662 P. vulgaris ATCC 13315 ND ND >128 >128 >128 ND 32 ND >128S. aureus ATCC 29213 ND ND 2 16 1 ND 0.5 ND 1 S. aureus MRSA13 416 >128 >128 32 32 8 16 64 S. aureus MRSA17 2 4 32 >128 8 4 2 2 16 S.aureus MRSA9 1 2 8 128 4 2 2 2 8 S. aureus SA206 ND ND 128 >128 16 ND 4ND 32 S. marcescens SM76 ND ND >128 >128 >128 ND >128 ND >128 S.marcescens SM82 >128 >128 >128 >128 >128 >128 >128 >128 >128 S.pneumoniae 406LE8 >128 >128 >128 >128 128 >128 64 128 128 S. pneumoniae60120 64 >128 >128 >128 >128 >128 128 128 >128 S. pneumoniae ATCC 3264 >128 >128 64 128 64 64 128 49619 ND = Not determined

Time Kill Assay

Time kill curves are used to determine the antimicrobial activity ofcationic peptides over a time interval. Briefly, in this assay, asuspension of microorganisms equivalent to a 0.5 McFarland Standard isprepared in 0.9% saline. This suspension is then diluted such that whenadded to a total volume of 9 ml of cation-adjusted Mueller Hinton broth,the inoculum size is 1×10⁶ CFU/ml. An aliquot of 0.1 ml is removed fromeach tube at pre-determined intervals up to 24 hours, diluted in 0.9%saline and plated in triplicate to determine viable colony counts. Thenumber of bacteria remaining in each sample is plotted over time todetermine the rate of cationic peptide killing. Generally a three ormore log₁₀ reduction in bacterial counts in the antimicrobial suspensioncompared to the growth controls indicate an adequate bactericidalresponse.

As shown in FIGS. 3A-E, most of the peptides demonstrate a three or morelog₁₀ reduction in bacterial counts in the antimicrobial suspensioncompared to the growth controls, indicating that these peptides have metthe criteria for a bactericidal response.

Example 5 Assays to Measure Enhanced Activity of Antibiotic Agent andCationic Peptide Combinations Killing Curves

Time kill curves resulting from combination of cationic peptide andantibiotic agent are compared to that resulting from agent alone.

The assay is performed as described above except that duplicate tubesare set up for each concentration of the antibiotic agent alone and ofthe combination of antibiotic agent and cationic peptide. Synergy isdemonstrated by at least a 100-fold (2 log₁₀) increase in killing at 24hours by the antibiotic agent and cationic peptide combination comparedto the antibiotic agent alone. A time kill assay is shown in FIG. 3E forMBI 26 in combination with vancomycin against a bacterial strain. Thecombination of peptide and antibiotic agent gave greater killing thaneither peptide or antibiotic agent alone.

FIC Measurements

In this method, synergy is determined using the agarose dilutiontechnique. An array of plates or tubes, each containing a combination ofpeptide and antibiotic in a unique concentration mix, is inoculated withbacterial isolates. When performing solid phase assays, calcium andmagnesium supplemented Mueller Hinton broth is used in combination witha low EEO agarose as the bacterial growth medium. Broth dilution assayscan also be used to determine synergy. Synergy is determined forcationic peptides in combination with a number of conventionalantibiotic agents, for example, penicillins, cephalosporins,carbapenems, monobactams, aminoglycosides, macrolides, fluoroquinolones,nisin and lysozyme.

Synergy is expressed as a fractional inhibitory concentration (FIC),which is calculated according to the equation below. An FIC ≦0.5 isevidence of synergy. An additive response has an FIC value >0.5 and ≦1,while an indifferent response has an FIC value >1 and ≦2.

${FIC} = {\frac{{MIC}\left( {{peptide}\mspace{14mu} {in}\mspace{14mu} {combination}} \right)}{{MIC}\left( {{peptide}\mspace{14mu} {alone}} \right)} + \frac{{MIC}\left( {{antibiotic}\mspace{14mu} {in}\mspace{14mu} {combination}} \right)}{{MIC}\left( {{antibiotic}\mspace{14mu} {alone}} \right)}}$

Tables 15, 16 and 17 present combinations of cationic peptides andantibiotic agents that display an FIC value of less than or equal to 1.Although FIC is measured in vitro and synergy defined as an FIC of lessthan or equal to 0.5, an additive effect may be therapeutically useful.As shown below, although all the microorganisms are susceptible (NCCLSbreakpoint definitions) to the tested antibiotic agents, the addition ofthe cationic peptide improves the efficacy of the antibiotic agent.

TABLE 15 Micro- Pep- organism Strain Antibiotic FIC tide S. aureus SA014Ciprofloxacin 0.63 MBI 26 S. aureus SA014 Ciprofloxacin 0.75 MBI 28 S.aureus SA014 Ciprofloxacin 1.00 MBI 11A2CN S. aureus SA093 Ciprofloxacin0.75 MBI 11A2CN S. aureus SA7609 Clindamycin 0.25 MBI 26 S. aureusSA7609 Methicillin 0.56 MBI 26 S. aureus SA7610 Clindamycin 0.63 MBI 26S. aureus SA7610 Methicillin 0.31 MBI 26 S. aureus SA7795 Ampicillin0.52 MBI 26 S. aureus SA7795 Clindamycin 0.53 MBI 26 S. aureus SA7796Ampicillin 1.00 MBI 26 S. aureus SA7796 Clindamycin 0.51 MBI 26 S.aureus SA7817 Ampicillin 0.50 MBI 26 S. aureus SA7818 Ampicillin 1.00MBI 26 S. aureus SA7818 Erythromycin 0.15 MBI 26 S. aureus SA7818Erythromycin 0.15 MBI 26 S. aureus SA7821 Erythromycin 0.50 MBI 26 S.aureus SA7821 Erythromycin 0.50 MBI 26 S. aureus SA7822 Ampicillin 0.25MBI 26 S. aureus SA7823 Ampicillin 0.25 MBI 26 S. aureus SA7824Ampicillin 1.00 MBI 26 S. aureus SA7825 Ampicillin 1.00 MBI 26 S. aureusSA7825 Erythromycin 1.00 MBI 26 S. aureus SA7825 Erythromycin 1.00 MBI26 S. aureus SA7834 Ampicillin 0.53 MBI 26 S. aureus SA7834 Clindamycin0.56 MBI 26 S. aureus SA7835 Ampicillin 0.53 MBI 26 S. aureus SA7836Ampicillin 0.75 MBI 26 S. aureus SA7837 Ampicillin 1.00 MBI 26 S. aureusSAATCC25293 Methicillin 0.50 MBI 26 S. aureus SAATCC29213 Methicillin0.31 MBI 26 S. aureus SAW1133 Methicillin 0.75 MBI 26 S. epidermidisSE8406 Clindamycin 0.50 MBI 26 S. epidermidis SE8416 Ampicillin 0.52 MBI31 S. epidermidis SE8416 Clindamycin 0.56 MBI 26 S. epidermidis SE8505Ampicillin 1.00 MBI 26 S. epidermidis SE8565 Ampicillin 1.00 MBI 26 S.epidermidis SH8575 Ampicillin 0.27 MBI 31 S. haemolyticus SA7797Ampicillin 0.50 MBI 31 S. haemolyticus SA7817 Ampicillin 0.26 MBI 31 S.haemolyticus SA7818 Ampicillin 0.52 MBI 31 S. haemolyticus SA7834Ampicillin 0.52 MBI 31 S. haemolyticus SA7835 Ampicillin 0.50 MBI 31 S.haemolyticus SH8459 Ampicillin 0.52 MBI 26 S. haemolyticus SH8472Ampicillin 0.56 MBI 26 S. haemolyticus SH8563 Ampicillin 0.75 MBI 26 S.haemolyticus SH8564 Ampicillin 0.62 MBI 26 S. haemolyticus SH8575Ampicillin 0.75 MBI 26 S. haemolyticus SH8576 Ampicillin 0.62 MBI 26 S.haemolyticus SH8578 Ampicillin 1.00 MBI 26 S. haemolyticus SH8597Ampicillin 1.00 MBI 31

TABLE 16 Teicoplanin (μg/ml) MBI 26 (μg/ml) Microorganism Strain Alone +MBI 26 Alone + Teicoplanin E. faecium 97001 VanB 0.5 0.25 64 4 E.faecium 97002 VanB 0.5 0.25 64 1 E. faecium 97003 VanB 0.5 0.25 64 1 E.faecium 97005 VanB 1 0.25 64 2 E. faecium 97006 VanB 0.5 0.5 64 4 E.faecium 97007 VanB 0.5 0.25 64 1 E. faecium 97008 VanB 0.5 0.25 64 4 E.faecium 97009 VanB 0.5 0.25 32 1 E. faecium 97010 VanB 0.5 0.25 64 4 E.faecium 97011 VanB 0.5 0.25 64 4 E. faecium 97012 VanB 8 0.25 64 4 E.faecium 97013 VanB 8 0.25 64 8 E. faecium 97014 VanB 8 0.25 32 4 E.faecium 97015 VanB 0.5 0.25 64 4 E. faecium 97016 VanB 0.5 0.25 64 4 E.faecalis 97040 VanB 0.5 0.25 64 8 E. faecalis 97041 VanB 1 0.25 64 8 E.faecalis 97042 VanB 1 0.25 64 8 E. faecalis 97043 VanB 0.5 0.25 64 8

TABLE 17 1. Amikacin Amikacin MIC (μg/ml) Peptide MIC (μg/ml) PeptideOrganism FIC Alone +Peptide Alone +Amikacin MBI 11B16CN A. baumanniiABI001 0.50 32 0.125 32 16 A. baumannii ABI003 0.53 16 0.5 16 8 P.aeruginosa PA022 0.38 64 8 64 16 P. aeruginosa PA037 0.25 16 2 >128 32S. maltophilia SMA018 0.31 128 8 32 8 S. maltophilia SMA022 0.09 >1288 >128 16 E. faecalis EFS008 0.28 32 8 8 0.25 MBI 21A2 A. baumanniiABI001 0.52 64 32 8 0.125 A. baumannii ABI003 0.52 16 8 8 4 P.aeruginosa PA022 0.50 64 16 8 2 S. maltophilia SMA018 0.50 >128 64 16 4S. maltophilia SMA022 0.25 >128 32 >128 32 E. faecium EFM004 0.56 12864 >128 16 E. faecalis EFS008 0.50 64 32 >128 0.125 S. aureus SA025 MRSA0.56 32 2 2 1 S. epidermidis SE003 0.38 32 4 >128 64 26 A. baumanniiABI001 0.50 32 8 8 2 A. baumannii ABI003 0.38 16 2 8 2 S. maltophiliaSMA021 0.13 128 8 32 2 S. maltophilia SMA037 0.19 128 16 >128 16 27 A.baumannii ABI003 0.52 16 0.25 8 4 B. cepacia BC005 0.50 64 16 >128 64 S.maltophilia SMA037 0.31 64 4 64 16 S. maltophilia SMA060 0.50 >128 0.12516 8 E. faecalis EFS008 0.53 32 1 4 2 MBI 29A3 B. cepacia BC003 0.50 328 >128 64 B. cepacia BC005 0.38 128 32 >128 32 S. maltophilia SMA0360.38 >128 32 64 16 S. maltophilia SMA063 0.56 >128 16 8 4 S. maltophiliaSMA064 0.56 >128 16 8 4 E. faecium EFM004 0.56 128 8 8 4 MBI 29F1 A.baumannii ABI001 0.51 32 0.25 8 4 A. baumannii ABI003 0.63 16 2 4 2 E.coli ECO022 0.51 16 0.125 4 2 P. aeruginosa PA022 0.53 128 64 4 0.125 S.maltophilia SMA021 0.31 128 8 8 2 S. maltophilia SMA022 0.31 >128 16 164 E. faecium EFM004 0.38 >128 32 32 8 E. faecalis EFS008 0.28 64 16 40.125 S. aureus SA014 MRSA 0.53 32 16 4 0.125 S. epidermidis SE002 0.3864 16 32 4 S. epidermidis SE003 0.50 64 16 32 8 2. CeftriaxoneCeftriaxone MIC (μg/ml) Peptide MIC (μg/ml) Peptide Organism FIC Alone+Peptide Alone +Ceftriaxone MBI 11B7CN A. baumannii ABI002 0.50 32 8 328 A. baumannii ABI006 0.25 128 16 32 4 B. cepacia BC003 0.52 32 16 >1284 P. aeruginosa PA008 0.25 128 16 128 16 P. aeruginosa PA024 0.50 6432 >128 0.125 S. maltophilia SMA020 0.75 >128 64 16 8 S. maltophiliaSMA021 0.50 >128 64 32 8 S. maltophilia SMA023 0.38 128 32 128 16 MBI11J02CN A. baumannii ABI005 0.56 16 8 8 0.5 B. cepacia BC003 0.50 164 >128 64 E. cloacae ECL014 0.38 128 16 32 8 E. cloacae ECL015 0.50 6416 32 8 P. aeruginosa PA008 0.50 64 0.125 64 32 P. aeruginosa PA039 0.5064 16 64 16 S. aureus SA025 MRSA 0.52 8 0.125 2 1 S. epidermidis SE0120.50 64 16 4 1 S. epidermidis SE073 0.38 128 16 4 1 26 A. baumanniiABI002 0.50 64 16 8 2 A. baumannii ABI005 0.56 16 8 2 0.125 B. cepaciaBC003 0.50 16 8 >128 0.125 E. cloacae ECL014 0.50 128 32 8 2 E. cloacaeECL015 0.19 64 4 32 4 K. pneumoniae KP003 0.56 8 4 16 1 P. aeruginosaPA008 0.13 64 8 128 0.125 P. aeruginosa PA024 0.50 16 4 128 32 S.maltophilia SMA019 0.50 >128 64 4 1 S. maltophilia SMA020 0.38 >128 32 41 S. aureus SA025 MRSA 0.52 8 0.125 1 0.5 S. epidermidis SE007 0.27 8 232 0.5 S. epidermidis SE012 0.27 64 16 64 1 3. CiprofloxacinCiprofloxacin MIC (μg/ml) Peptide MIC (μg/ml) Peptide Organism FIC Alone+Peptide Alone +Ciprofloxacin MBI 11A1CN S. aureus SA10 0.53 32 16 128 4S. aureus SA11 0.50 64 32 >128 1 MBI 11D18CN P. aeruginosa PA24 0.31 164 >128 16 P. aeruginosa PA77 0.50 2 0.5 128 32 MBI 21A1 S. aureus SA250.16 4 0.125 32 4 S. aureus SA93 0.50 32 8 4 1 P. aeruginosa PA4 0.500.5 0.125 128 32 P. aeruginosa PA41 0.50 4 1 16 4 MBI 21A2 S. aureusSA25 0.50 2 0.5 16 4 S. aureus SA93 0.38 32 8 16 2 P. aeruginosa PA40.50 0.5 0.125 >128 64 P. aeruginosa PA41 0.50 4 1 64 16 MBI 26 S.aureus SA11 0.50 64 32 128 0.125 P. aeruginosa PA41 0.50 4 1 128 32 P.aeruginosa PA77 0.56 2 0.125 128 64 A. calcoaceticus 1 0.51 0.5 0.25 >641 A. calcoaceticus 6 0.50 1 0.25 >32 16 E. cloacae 13 0.27 1 0.25 >128 4E. cloacae 15 0.38 1 0.25 >32 8 E. cloacae 16 0.38 2 0.25 >32 16 P.aeruginosa 23 0.53 1 0.5 >32 2 P. aeruginosa 24 0.53 1 0.5 >32 2 S.maltophilia 34 0.25 2 0.25 >32 8 S. maltophilia 35 0.50 2 0.5 >32 16 MBI27 S. aureus SA10 0.75 32 8 2 1 S. aureus SA93 0.63 32 4 2 1 P.aeruginosa PA4 0.75 0.5 0.25 32 8 MBI 28 S. aureus SA11 0.63 32 16 64 8S. aureus SA25 0.56 2 0.125 2 1 P. aeruginosa PA24 0.75 32 8 64 32 29 S.aureus SA10 0.38 32 4 4 1 S. aureus SA93 0.50 32 8 2 0.5 P. aeruginosaPA41 0.52 8 4 8 0.125 P. aeruginosa PA77 0.50 2 0.5 64 16 A.calcoaceticus 5 0.56 2 1 16 1 A. calcoaceticus 9 0.56 2 1 16 1 E.cloacae 14 0.50 1 0.25 >16 8 E. cloacae 15 0.50 1 0.25 >16 8 P.aeruginosa 30 0.56 4 0.25 >16 16 P. aeruginosa 31 0.53 16 0.5 >16 16 S.maltophilia 34 0.27 2 0.5 >16 0.5 S. maltophilia 35 0.63 2 0.25 >16 16S. maltophilia 36 0.56 8 0.5 >16 16 MBI 29A2 S. aureus SA10 0.52 32 0.54 2 S. aureus SA93 0.50 32 8 2 0.5 P. aeruginosa PA24 0.63 32 16 64 8MBI 29A3 S. aureus SA10 0.75 32 16 2 0.5 S. aureus SA25 0.63 4 2 1 0.125P. aeruginosa PA24 0.50 32 16 64 0.125 P. aeruginosa PA41 0.63 4 0.5 8 44. Gentamicin Gentamicin MIC (μg/ml) Peptide MIC (μg/ml) PeptideOrganism FIC Alone +Peptide Alone +Gentamicin MBI 11A1CN S. maltophilia0.31 8 2 >128 16 SMA019 S. maltophilia 0.31 8 2 >128 16 SMA020 E.faecium EFM004 0.28 >128 64 32 1 S. aureus SA014 0.56 32 2 8 4 MRSA S.epidermidis SE074 0.51 128 1 32 16 MBI 11B16CN A. baumannii ABI001 0.3164 4 16 4 A. baumannii ABI002 0.31 32 2 16 4 A. calcoaceticus 0.25 8 132 4 AC001 P. aeruginosa PA022 0.38 32 8 64 8 P. aeruginosa PA041 0.31 82 >128 16 S. maltophilia 0.31 >128 64 >128 16 SMA016 S. maltophilia 0.3864 8 32 8 SMA019 E. faecalis EFS008 0.38 >128 64 4 0.5 S. aureus SA0140.53 32 1 8 4 MRSA MBI 11D18CN A. baumannii ABI001 0.27 64 16 32 0.5 A.baumannii ABI002 0.56 16 8 32 2 E. coli ECO006 0.27 64 16 8 0.125 K.pneumonia KP020 0.50 64 32 32 0.125 P. aeruginosa PA022 0.52 16 8 80.125 P. aeruginosa PA041 0.14 8 0.125 64 8 S. maltophilia 0.38 128 1664 16 SMA016 S. maltophilia 0.19 32 4 8 0.5 SMA019 E. faecium EFM0040.05 >128 8 8 0.125 E. faecalis EFS008 0.19 128 8 2 0.25 S. aureus SA0140.13 32 2 2 0.125 MRSA S. aureus SA025 0.14 64 1 1 0.125 MRSA S.epidermidis SE071 0.27 16 4 8 0.125 S. epidermidis SE074 0.09 64 4 40.125 MBI 21A2 A. baumannii ABI002 0.56 32 16 8 0.5 P. aeruginosa PA0220.50 32 8 8 2 S. maltophilia 0.50 64 16 16 4 SMA019 S. maltophilia 0.5064 16 16 4 SMA020 S. maltophilia 0.50 64 16 16 4 SMA021 S. aureus SA0250.63 64 32 8 1 MRSA MBI 26 A. baumannii ABI001 0.50 64 16 8 2 A.baumannii ABI002 0.53 16 0.5 8 4 P. aeruginosa PA041 0.63 8 1 64 32 S.maltophilia 0.25 >128 32 >128 32 SMA016 S. maltophilia 0.38 64 16 16 2SMA017 MBI 27 A. baumannii ABI002 0.52 32 0.5 8 4 P. aeruginosa PA0220.52 32 16 8 0.125 S. maltophilia 0.50 >128 64 64 16 SMA016 S.maltophilia 0.52 128 64 8 0.125 SMA017 E. faecalis EFS008 0.38 >128 64 40.5 S. aureus SA014 0.50 32 0.125 2 1 MRSA MBI 29 S. maltophilia 0.53 3216 4 0.125 SMA019 S. maltophilia 0.53 32 16 4 0.125 SMA020 E. faecalisEFS008 0.38 128 32 1 0.125 S. epidermidis SE074 0.50 128 0.5 4 2 MBI29A3 S. maltophilia 0.31 64 16 2 0.125 SMA019 S. maltophilia 0.31 64 162 0.125 SMA021 MBI 29F1 P. aeruginosa PA023 0.52 8 0.125 128 64 S.maltophilia 0.56 >128 16 32 16 SMA016 S. maltophilia 0.53 64 32 4 0.125SMA017 Deber A2KA2 A. baumannii ABI001 0.53 64 32 >128 8 A. baumanniiABI002 0.50 64 32 >128 0.125 A. calcoaceticus 0.56 8 4 >128 16 AC001 P.aeruginosa PA022 0.52 32 16 >128 4 P. aeruginosa PA041 0.50 16 8 >1280.125 S. maltophilia 0.50 128 64 >128 0.125 SMA017 S. maltophilia 0.50128 64 >128 0.125 SMA020 5. Mupirocin Mupirocin MIC (μg/ml) Peptide MIC(μg/ml) Peptide Organism FIC Alone +Peptide Alone +Mupirocin MBI 11A1CNE. coli SBECO2 0.05 >100 30 128 2 E. coli ECO1 0.14 >100 10 32 4 MBI11A3CN E. coli SBECO1 0.43 100 30 64 8 MBI 11B4CN E. coli SBECO1 0.36100 30 8 0.5 E. coli SBECO2 0.09 >100 30 32 2 MBI 11D18CN E. coli SBECO10.36 100 30 2 0.125 E. coli SBECO2 0.06 >100 30 16 0.5 P. aeruginosaSBPA1 0.35 >100 100 128 32 P. aeruginosa PA4 0.53 >100 30 128 64 S.marcescens 0.16 >100 100 >128 16 SBSM1 S. marcescens 0.35 >100 100 >12864 SBSM2 MBI 11G13CN E. coli SBECO2 0.16 >100 30 64 8 E. coli ECO5 0.43100 30 64 8 MBI 21A1 E. coli SBECO2 0.28 >100 30 8 2 E. coli ECO3 0.28100 3 8 2 P. aeruginosa SBPA1 0.53 >100 30 64 32 MBI 26 E. coli SBECO20.16 >100 30 8 1 E. coli ECO5 0.43 100 30 8 1 P. aeruginosa PA20.51 >100 10 128 64 P. aeruginosa PA4 0.23 >100 100 >128 32 S. aureusSBSA4 0.28 >100 30 32 8 MBI 27 E. coli SBECO2 0.51 >100 10 4 2 P.aeruginosa PA2 0.25 >100 0.1 64 16 P. aeruginosa PA4 0.50 >100 0.3 32 16S. aureus SBSA3 0.23 100 10 16 2 S. aureus SBSA4 0.50 >100 0.3 4 2 MBI28 E. coli SBECO1 0.50 100 0.1 4 2 E. coli ECO2 0.33 100 30 4 0.125 P.aeruginosa SBPA1 0.53 >100 30 32 16 P. aeruginosa PA4 0.50 >100 3 32 16S. aureus SBSA4 0.51 >100 10 4 2 MBI 29 S. marcescens 0.23 >100 100 >12832 SBSM1 S. aureus SBSA3 0.35 100 10 16 4 S. aureus SBSA4 0.51 >100 10 42 MBI 29A3 P. aeruginosa PA2 0.50 >100 0.1 32 16 P. aeruginosa PA30.50 >100 0.1 16 8 S. marcescens 0.16 >100 100 >128 16 SBSM1 S.marcescens 0.35 >100 100 >128 64 SBSM2 6. Piperacillin Piperacillin MIC(μg/ml) Peptide MIC (μg/ml) Peptide Organism FIC Alone +Peptide Alone+Piperacillin MBI 11B7CN E. cloacae 6 0.56 >128 16 32 16 E. cloacae 90.50 >128 1 32 16 E. cloacae 10 0.50 >128 0.5 32 16 S. maltophilia 50.50 >128 64 >128 64 S. maltophilia 9 0.50 >128 64 >128 64 S.maltophilia 11 0.38 >128 64 >128 32 S. marcescens 1 0.27 32 8 >128 4 P.aeruginosa 23 0.56 32 2 128 64 H. influenzae 1 0.50 64 32 >128 0.125 H.influenzae SB1 0.50 0.5 0.25 >128 0.125 S. aureus 19 MRSA 0.50 128 32 41 MBI 11B9CN A. calcoaceticus 3 0.56 64 32 32 2 S. maltophilia 50.50 >128 64 >128 64 S. maltophilia 13 0.38 >128 64 >128 32 S.marcescens SB1 0.26 64 16 >128 2 P. aeruginosa 15 0.50 >128 64 >128 64P. aeruginosa 23 0.13 128 16 64 0.5 H. influenzae 3 0.50 0.5 0.25 >1280.125 H. influenzae SB1 0.50 0.5 0.25 >128 0.125 S. aureus 19 MRSA 0.38128 16 4 1 S. aureus 0.56 128 8 2 1 SB2MRSA MBI 11CN P. aeruginosa 220.52 >128 4 64 32 P. aeruginosa 23 0.53 128 64 128 4 S. aureus 18 MRSA0.50 >128 0.5 32 16 S. aureus 19 MRSA 0.38 >128 64 8 1 MBI 11D18CN A.calcoaceticus 3 0.38 64 8 32 8 E. cloacae 9 0.31 >128 16 64 16 E.cloacae 10 0.50 >128 64 32 8 S. maltophilia 2 0.50 64 16 32 8 S.marcescens 1 0.14 64 8 >128 4 P. aeruginosa 23 0.38 128 32 64 8 P.aeruginosa 41 0.56 64 32 >128 16 H. influenzae 3 0.53 0.5 0.25 >128 8 H.influenzae SB1 0.52 0.5 0.25 >128 4 S. aureus 19 MRSA 0.38 128 16 4 1 S.aureus 0.50 128 32 2 0.5 SB2MRSA MBI 11E3CN S. maltophilia 11 0.51 >1282 128 64 S. marcescens SB1 0.26 64 16 >128 2 P. aeruginosa 23 0.27 12832 64 1 P. aeruginosa 32 0.63 64 32 64 8 H. influenzae 1 0.52 64 32 >1284 H. influenzae 2 0.31 32 8 >128 16 S. aureus 19 MRSA 0.50 >128 64 4 1MBI 11F3CN P. aeruginosa 23 0.51 128 64 64 0.5 P. aeruginosa 41 0.63 324 128 64 S. aureus 19 MRSA 0.38 >128 32 4 1 S. aureus 0.50 >128 64 8 2SB3MRSA MBI 11F4CN E. cloacae 10 0.52 >128 4 16 8 S. maltophilia 2 0.5364 32 16 0.5 S. marcescens 1 0.25 >128 64 >128 0.5 P. aeruginosa 70.38 >128 64 64 8 P. aeruginosa 23 0.31 >128 64 64 4 H. influenzae SB10.50 0.5 0.25 >128 0.125 S. aureus 19 MRSA 0.53 128 4 4 2 MBI 11G7CN A.calcoaceticus 3 0.50 128 32 64 16 S. marcescens 1 0.25 64 16 >128 1 P.aeruginosa 7 0.50 >128 64 >128 64 P. aeruginosa 23 0.50 128 64 >128 1 H.influenzae SB1 0.52 0.5 0.25 >128 4 S. aureus 18 MRSA 0.50 >128 64 32 8S. aureus 19 MRSA 0.56 128 64 8 0.5 MBI 21A2 E. coli 1 0.53 >128 8 4 2S. maltophilia 6 0.38 >128 64 128 16 S. maltophilia 14 0.53 128 4 32 16S. marcescens 1 0.27 64 16 >128 4 P. aeruginosa 23 0.19 64 8 >128 16 H.influenzae 1 0.31 64 4 >128 64 H. influenzae 2 0.38 128 32 >128 32 S.aureus 19 MRSA 0.51 128 64 >128 2 S. aureus 0.56 128 64 32 2 SB2MRSA MBI26 S. maltophilia 3 0.50 128 32 16 4 S. marcescens 1 0.50 64 32 >128 0.5P. aeruginosa 7 0.25 >128 32 >128 32 P. aeruginosa 41 0.53 64 32 128 4H. influenzae 1 0.53 64 32 >128 8 H. influenzae 2 0.51 128 64 >128 2 S.aureus 19 MRSA 0.16 128 16 32 1 S. aureus 0.31 128 64 >128 16 SB3MRSA A.calcoaceticus 7 0.25 32 4 >32 8 A. calcoaceticus 8 0.19 64 4 >32 8 E.cloacae 13 0.16 128 4 >32 8 P. aeruginosa 23 0.27 256 4 >64 32 P.aeruginosa 28 0.14 >512 16 >128 32 S. maltophilia 34 0.25 >512 4 >32 16S. maltophilia 35 0.26 >256 4 >32 16 MBI 29 S. marcescens 1 0.14 6432 >128 4 P. aeruginosa 7 0.53 128 4 16 8 P. aeruginosa 23 0.50 128 3216 4 P. aeruginosa 41 0.56 64 32 64 4 H. influenzae 1 0.51 32 16 160.125 S. aureus 11 MRSA 0.50 >128 0.5 16 8 A. calcoaceticus 2 0.50 >5124 16 8 A. calcoaceticus 7 0.25 32 4 >16 4 E. cloacae 16 0.50 >512 4 >1616 E. cloacae 17 0.50 >512 4 >16 16 P. aeruginosa 28 0.13 >512 8 >64 16P. aeruginosa 29 0.27 512 8 >32 16 S. maltophilia 34 0.25 >512 4 >16 8S. maltophilia 38 0.28 >512 32 >32 16 S. maltophilia 40 0.25 >512 4 >3216 S. maltophilia 42 0.25 >512 4 >16 8 7. Tobramycin Tobramycin MIC(μg/ml) Peptide MIC (μg/ml) Peptide Organism FIC Alone +Peptide Alone+Tobramycin MBI 11A1CN P. aeruginosa PA026 0.50 8 4 >128 0.125 P.aeruginosa PA032 0.50 16 8 >128 0.5 S. maltophilia SMA029 0.16 1284 >128 32 S. maltophilia SMA030 0.27 128 2 >128 64 S. aureus SA0140.50 >128 0.125 16 8 S. aureus SA025 0.50 >128 0.125 8 4 S. haemolyticus0.52 4 2 8 0.125 SHA001 S. haemolyticus 0.51 8 4 16 0.125 SHA005 MBI11B9CN A. baumannii ABI001 0.50 16 4 32 8 B. cepacia BC002 0.38 >12864 >128 32 P. aeruginosa PA008 0.50 32 0.125 128 64 P. aeruginosa PA0250.56 32 2 128 64 S. maltophilia SMA029 0.13 64 4 >128 16 MBI 11CN A.baumannii ABI001 0.50 16 4 64 16 E. coli ECO006 0.53 8 4 8 0.25 P.aeruginosa PA032 0.52 16 8 >128 4 S. maltophilia SMA029 0.51 128 64 >1282 S. maltophilia SMA035 0.38 32 4 128 32 MBI 11D18CN A. baumannii ABI0010.31 16 4 64 4 A. baumannii ABI002 0.53 8 4 16 0.5 S. maltophilia SMA0270.19 32 4 >128 16 S. maltophilia SMA029 0.16 128 4 32 4 S. aureus SA018MRSA 0.56 64 4 32 16 S. haemolyticus 0.53 4 0.125 2 1 SHA001 MBI 11F3CNA. baumannii ABI001 0.53 16 0.5 32 16 A. baumannii ABI002 1.00 4 2 16 8P. aeruginosa PA032 0.50 16 4 >128 64 S. maltophilia SMA029 0.28 128 32128 4 S. maltophilia SMA030 0.26 128 1 128 32 S. aureus SA014 MRSA0.51 >128 2 4 2 S. haemolyticus 0.56 4 0.25 4 2 SHA005 MBI 11G13CN A.baumannii ABI001 0.50 16 4 128 32 P. aeruginosa PA022 0.56 8 4 >128 16S. maltophilia SMA029 0.50 128 64 >128 0.125 S. maltophilia SMA030 0.50128 64 >128 0.125 S. aureus SA025 MRSA 0.50 >128 0.125 4 2 MBI 21A1 B.cepacia BC001 0.25 128 32 >128 0.25 P. aeruginosa PA022 0.53 8 4 4 0.125P. aeruginosa PA026 0.51 8 4 16 0.125 S. maltophilia SMA029 0.28 128 4128 32 S. maltophilia SMA030 0.16 128 4 >128 32 S. aureus SA014 MRSA0.50 >128 0.125 32 16 S. aureus SA025 MRSA 0.50 >128 0.125 2 1 S.haemolyticus 0.50 2 0.5 16 4 SHA001 S. haemolyticus 0.38 4 1 32 4 SHA005MBI 22A1 S. maltophilia SMA030 0.26 128 1 32 8 S. maltophilia SMA0310.25 128 0.5 32 8 S. aureus SA014 MRSA 0.27 >128 4 8 2 S. epidermidisSE072 0.50 >128 0.125 16 8 S. epidermidis SE073 0.50 >128 0.125 16 8 S.epidermidis SE080 0.56 32 16 2 0.125 MBI 26 S. maltophilia SMA029 0.05128 4 >128 4 S. maltophilia SMA030 0.05 128 4 >128 4 S. epidermidisSE067 0.38 >128 64 2 0.25 S. epidermidis SE068 0.27 >128 4 2 0.5 MBI 27E. coli ECO006 0.56 8 0.5 8 4 S. maltophilia SMA029 0.50 64 16 16 4 S.maltophilia SMA031 0.53 128 4 16 8 MBI 29 A. baumannii ABI001 0.53 16 84 0.125 E. coli ECO004 0.53 2 1 4 0.125 E. coli ECO006 0.53 8 4 4 0.125K. pneumoniae KP008 0.52 0.5 0.25 8 0.125 P. aeruginosa PA030 0.52 16 88 0.125 S. maltophilia SMA031 0.50 >128 0.25 16 8 S. maltophilia SMA0320.53 128 4 16 8 S. epidermidis SE072 0.53 >128 8 16 8 MBI 29A3 P.aeruginosa PA022 0.56 8 4 4 0.25 P. aeruginosa PA028 0.50 32 16 32 0.125P. aeruginosa PA029 0.51 32 16 16 0.125 S. maltophilia SMA029 0.28 128 416 4 S. maltophilia SMA030 0.28 128 4 16 4 REWH S. maltophilia SMA0290.08 128 2 >128 16 53A5CN S. maltophilia SMA030 0.13 128 0.25 >128 32 S.aureus SA014 MRSA 0.50 >128 0.125 16 8 8. Vancomycin Vancomycin MIC(μg/ml) Peptide MIC (μg/ml) Peptide Organism FIC Alone +Peptide Alone+Vancomycin MBI 11A1CN E. faecalis EFS001 0.53 1 0.5 4 0.125 E. faecalisEFS006 0.50 8 4 128 0.25 E. faecalis EFS007 0.50 4 2 128 0.5 E. faecalisEFS010 0.27 16 4 128 2 E. faecalis EFS012 0.25 >128 32 64 8 E. faecalisEFS014 0.51 128 1 4 2 E. faecium EFM004 0.50 >128 0.5 32 16 E. faeciumEFM007 0.28 128 4 64 16 E. faecium EFM009 0.25 32 4 64 8 MBI 11D18CN E.faecalis EFS001 0.38 1 0.125 8 2 E. faecalis EFS004 0.50 2 0.5 8 2 E.faecalis EFS011 0.50 64 32 64 0.125 E. faecalis EFS012 0.38 >128 64 16 2E. faecalis EFS014 0.16 128 4 4 0.5 E. faecium EFM004 0.50 >128 64 8 2E. faecium EFM009 0.52 64 32 8 0.125 E. faecium EFM010 0.28 >128 64 80.25 E. faecium EFM011 0.50 >128 64 8 2 MBI 21A1 E. faecalis EFS007 0.562 1 16 1 E. faecalis EFS012 0.16 128 16 32 1 E. faecalis EFS013 0.28 12832 32 1 E. faecium EFM010 0.56 64 32 32 2 MBI 26 E. faecalis EFS005 0.3116 4 >128 16 E. faecalis EFS012 0.07 >128 2 16 1 E. faecalis EFS0130.07 >128 2 16 1 E. faecium EFM010 0.31 32 2 32 8 E. faecium EFM011 0.3132 2 32 8 E. faecium EFM012 0.31 32 2 64 16 E. faecium EFM014 0.27 >1284 32 8 E. faecium EFM016 0.51 128 1 8 4 MBI 29 E. faecalis EFS005 0.3816 4 32 4 E. faecalis EFS010 0.38 64 16 2 0.25 E. faecalis EFS0120.50 >128 64 2 0.5 E. faecium EFM005 0.53 128 4 4 E. faecium EFM016 0.51128 1 4 2 MBI 29A3 E. faecalis EFS003 0.56 4 2 32 2 E. faecalis EFS0050.28 16 4 32 1 E. faecalis EFS011 0.50 16 4 32 8 E. faecalis EFS014 0.5264 1 1 0.5 E. faecium EFM006 0.52 >128 4 4 2

Example 6 Overcoming Tolerance by Administering a Combination ofAntibiotic Agent and Cationic Peptide

Tolerance to an antibiotic agent is associated with a defect inbacterial cellular autolytic enzymes such that an antimicrobial agent isbacteriostatic rather than bactericidal. Tolerance is indicated when aratio of minimum bactericidal concentration (MBC) to minimum inhibitoryconcentration (MIC) (MBC:MIC) is ≧32.

The agarose dilution assay is adapted to provide both the MBC and MICfor an antimicrobial agent alone and an agent in combination with apeptide. Following determination of MIC, MBC is determined from theagarose dilution assay plates by swabbing the inocula on plates at andabove the MIC and resuspending the swab in 1.0 ml of saline. A 0.01 mlaliquot is plated on agarose medium (subculture plates) and theresulting colonies are counted. If the number of colonies is less than0.1% of the initial inoculum (as determined by a plate count immediatelyafter inoculation of the MIC test plates), then ≧99.9% killing hasoccurred. The MBC end point is defined as the lowest concentration ofthe antimicrobial agent that kills 99.9% of the test bacteria.

Thus, tolerance of a microorganism to an antimicrobial agent occurs whenthe number of colonies growing on subculture plates exceeds the 0.1%cutoff for several successive concentrations above the observed MIC. Acombination of antimicrobial agent and cationic peptide that breakstolerance results in a decrease in the MBC:MIC ratio to <32. Table 18shows that the combination of Vancomycin and MBI 26 overcomes thetolerance of the organisms listed.

TABLE 18 Vancomycin MIC MBC Vancomycin + MBI 26 (μg/ (μg/ MBC/ MIC MBCMBC/ Organism ml) ml) MIC (μg/ml) (μg/ml) MIC E. casseliflavus2 >128 >64 0.5 2 4 ECA001 E. faecium EFM001 0.5 >128 >256 0.5 0.5 1 E.faecium EFM020 1 >128 >128 0.5 4 8 E. faecalis EFS001 1 >128 >128 0.5 48 E. faecalis EFS004 1 >128 >128 1 2 2 E. faecalis EFS007 4 128 32 2 2 1E. faecalis EFS009 4 >128 >32 4 4 1 E. faecalis EFS015 1 >128 >128 0.50.5 1

Example 7 Overcoming Inherent Resistance by Administering a Combinationof Antibiotic Agent and Cationic Peptide

Peptides are tested for their ability to overcome the inherentantimicrobial resistance of microorganisms, including those encounteredin hospital settings, to specific antimicrobials. Overcoming resistanceis demonstrated when the antibiotic agent alone exhibits minimal or noactivity against the microorganism, but when used in combination with acationic peptide, results in susceptibility of the microorganism.

The agarose dilution assay described above is used to determine theminimum inhibitory concentration (MIC) of antimicrobial agents andcationic peptides, alone and in combination. Alternatively, the brothdilution assay or time kill curves can be used to determine MICs. Tables19, 20, 21 and 22 present MIC values for antibiotic agents alone and incombination with peptide at the concentration shown. In all cases, themicroorganism is inherently resistant to its mode of action, thus, theantibiotic agent is not effective against the test microorganism. Inaddition, the antibiotic agent is not clinically prescribed against thetest microorganism.

In the data presented below, the MIC values for the antibiotic agentswhen administered in combination with peptide are decreased, from equalto or above the resistant breakpoint to below it.

TABLE 19 Erythromycin MBI 26 MIC MIC (μg/ml) (μg/ml) MicroorganismAlone + MBI 26 Alone + Erythro. A. calcoaceticus AC001 32 1 16 8 K.pneumoniae KP001 32 0.25 16 8 K. pneumoniae KP002 256 0.5 64 32 P.aeruginosa PA041 128 4 64 32

TABLE 20 MBI 26 MIC Vancomycin (μg/ml) MIC (μg/ml) + Vanco-Microorganism Alone + MBI 26 Alone mycin E. gallinarum 97044 VanC 8 2 80.5 E. gallinarum 97046 VanC 32 1 2 4 E. gallinarum 97047 VanC 128 16 648 E. gallinarum 97048 VanC 32 4 2 2 E. gallinarum 97049 VanC 128 4 64 16E. casseliflavus 97056 VanC 8 2 8 1 E. casseliflavus 97057 VanC 4 2 20.5 E. casseliflavus 97058 VanC 2 1 4 0.25 E. casseliflavus 97059 VanC 42 32 0.5 E. casseliflavus 97060 VanC 2 2 0.5 0.25

TABLE 21 MBI 26 Teicoplanin MIC (μg/ml) MIC (μg/ml) + Vanco-Microorganism Alone + MBI 26 Alone mycin E. gallinarum 97044 VanC 0.50.25 64 1 E. gallinarum 97046 VanC 1 0.25 8 1 E. gallinarum 97047 VanC 80.25 64 32 E. gallinarum 97048 VanC 0.5 0.25 8 1 E. gallinarum 97049VanC 2 0.25 64 32 E. casseliflavus 97056 VanC 0.5 0.25 64 2 E.casseliflavus 97057 VanC 0.5 0.25 64 0.5 E. casseliflavus 97058 VanC 0.50.25 32 0.5 E. casseliflavus 97059 VanC 0.5 0.25 64 1 E. casseliflavus97060 VanC 0.5 0.25 64 1

TABLE 22 1. Amikacin Amikacin MIC (μg/ml) Peptide MIC (μg/ml) PeptideOrganism FIC Alone +Peptide Alone +Amikacin MBI 11B16CN A. baumanniiABI001 0.25 32 4 32 4 S. maltophilia SMA018 0.31 128 8 32 8 S.maltophilia SMA022 0.14 >128 4 >128 32 S. aureus SA014 MRSA 0.75 32 8 84 S. aureus SA025 MRSA 0.63 32 4 8 4 MBI 21A2 S. maltophilia SMA0180.53 >128 8 16 8 S. maltophilia SMA060 0.31 >128 16 >128 64 S. aureusSA025 MRSA 0.56 32 2 2 1 MBI 26 S. maltophilia SMA022 0.19 128 8 64 8 S.maltophilia SMA037 0.19 128 16 >128 16 MBI 27 A. baumannii ABI001 1.0032 16 8 4 B. cepacia BC005 0.50 64 16 >128 64 S. maltophilia SMA0360.56 >128 16 64 32 S. maltophilia SMA037 0.31 64 4 64 16 S. aureus SA025MRSA 0.75 32 16 2 0.5 MBI 29A3 B. cepacia BC003 0.63 32 16 >128 32 B.cepacia BC005 0.38 128 32 >128 32 S. maltophilia SMA036 0.53 >128 8 6432 S. maltophilia SMA063 0.56 >128 16 8 4 MBI 29F1 A. baumannii ABI0010.75 32 16 8 2 S. maltophilia SMA018 0.56 128 8 4 2 S. maltophiliaSMA021 0.31 128 8 8 2 S. aureus SA014 MRSA 0.53 32 16 4 0.125 S. aureusSA025 MRSA 0.63 32 16 1 0.125 Debar A2KA2 A. baumannii ABI001 0.63 3216 >128 32 S. aureus SA025 MRSA 0.50 32 0.125 16 8 2. CeftriaxoneCeftriaxone MIC (μg/ml) Peptide MIC (μg/ml) Peptide Organism FIC Alone+Peptide Alone +Ceftriaxone MBI 11B7CN P. aeruginosa PA008 0.50 1280.125 128 64 S. maltophilia SMA021 0.50 >128 1 32 16 S. maltophiliaSMA023 0.56 128 8 128 64 MBI 11J02CN P. aeruginosa PA008 0.50 64 0.12564 32 P. aeruginosa PA039 0.52 64 1 64 32 MBI 26 P. aeruginosa PA0080.13 64 8 128 0.125 P. aeruginosa PA024 0.50 16 4 128 32 S. maltophiliaSMA021 0.25 >128 1 8 2 3. Gentamicin Gentamicin MIC (μg/ml) Peptide MIC(μg/ml) Peptide Organism FIC Alone +Peptide Alone +Gentamicin MBI11B16CN S. aureus SA014 MRSA 0.53 32 1 8 4 MBI 27 S. aureus SA014 MRSA0.50 32 0.125 2 1 4. Mupirocin Mupirocin MIC (μg/ml) Peptide MIC (μg/ml)Peptide Organism FIC Alone +Peptide Alone +Mupirocin MBI 11B4CN E. coliECO3 0.53 100 3 16 8 MBI 11D18CN E. coli ECO3 0.26 100 1 4 1 MBI 21A1 E.coli ECO1 0.50 >100 3 2 1 E. coli ECO2 0.53 100 3 2 1 E. coli ECO3 0.28100 3 8 2 MBI 26 E. coli ECO1 0.50 >100 3 2 1 MBI 27 P. aeruginosa PA20.25 >100 0.1 64 16 P. aeruginosa PA4 0.50 >100 0.3 32 16 MBI 28 E. coliSBECO1 0.50 100 0.1 4 2 P. aeruginosa PA4 0.50 >100 3 32 16 MBI 29A3 P.aeruginosa SBPA2 0.50 >100 0.1 16 8 P. aeruginosa PA2 0.50 >100 0.1 3216 P. aeruginosa PA3 0.50 >100 0.1 16 8 P. aeruginosa PA4 0.50 >100 0.116 8 5. Piperacillin Piperacillin MIC (μg/ml) Peptide MIC (μg/ml)Peptide Organism FIC Alone +Peptide Alone +Piperacillin MBI 11B7CN S.aureus 19 MRSA 0.50 128 0.5 4 2 MBI 11D18CN S. aureus 19 MRSA 0.52 128 24 2 MBI 11E3CN S. aureus 19 MRSA 0.51 >128 2 4 2 MBI 11F3CN S. aureus 19MRSA 0.51 >128 2 4 2 S. aureus SB3MRSA 0.52 >128 4 8 4 MBI 11F4CN S.aureus 19 MRSA 0.53 128 4 4 2 MBI 11G7CN S. aureus 19 MRSA 0.25 128 0.58 2 MBI 21A2 S. aureus 19 MRSA 0.25 128 0.5 >128 64 MBI 26 S. aureus 19MRSA 0.13 128 0.5 32 4 MBI 29 S. aureus 18 MRSA 0.52 >128 4 16 8 6.Tobramycin Tobramycin MIC (μg/ml) Peptide MIC (μg/ml) Peptide OrganismFIC Alone +Peptide Alone +Tobramycin MBI 11A1CN S. aureus SA0140.50 >128 0.125 16 8 S. aureus SA025 0.50 >128 0.125 8 4 S. haemolyticusSHA005 0.51 8 4 16 0.125 MBI 11D18CN S. aureus SA018 MRSA 0.56 64 4 3216 MBI 11F3CN S. aureus SA014 MRSA 0.51 >128 2 4 2 MBI 11G13CN S. aureusSA025 MRSA 0.50 >128 0.125 4 2 MBI 21A1 S. aureus SA014 MRSA 0.50 >1280.125 32 16 S. aureus SA025 MRSA 0.50 >128 0.125 2 1 MBI 22A1 S. aureusSA014 MRSA 0.27 >128 4 8 2

Example 8 Overcoming Acquired Resistance by Administering a Combinationof Antibiotic Agent and Cationic Peptide

An antibiotic agent can become ineffective against a previouslysusceptible microorganism if the microorganism acquires resistance tothe agent. However, acquired resistance can be overcome when the agentis administered in combination with a cationic peptide. For examplevancomycin resistant enterococci (VRE) become susceptible to vancomycinwhen it is used in combination with a cationic peptide such as MBI 26.This combination is likely to be effective against other organismsacquiring resistance to vancomycin including but not limited to strainsof methicillin resistant S. aureus (MRSA).

Similarly teicoplanin resistant enterococci become susceptible toteicoplanin when teicoplanin is used in combination with cationicpeptides such as MBI 26.

As described previously, the agarose dilution assay is used to determinethe MIC for antibiotic agents administered alone and in combination withcationic peptide. Alternatively the broth dilution assay or time killcurves can be employed. Tables 23 and 25 presents results showing thatadministration of a cationic peptide in combination with an antibioticagent overcomes acquired resistance. Table 24 presents results showingadministration of MBI 26 in combination with teicoplanin againstteicoplanin resistant enterococci.

TABLE 23 MIC MIC alone comb. Peptide Microorganism Strain Antibioticagent (μg/ml) (μg/ml) Peptide MIC A. calcoaceticus 002 Tobramycin 8 1MBI 29 4 A. calcoaceticus 003 Ceftazidime 32 2 MBI 26 32 A.calcoaceticus 003 Ceftazidime 32 2 MBI 29 8 A. calcoaceticus 003Ciprofloxacin 8 1 MBI 29 16 A. calcoaceticus 004 Ciprofloxacin 8 4 MBI26 4 A. calcoaceticus 010 Ceftazidime 32 2 MBI 26 32 E. faecium ATCC29212 Mupirocin 100 0.1 MBI 11CN 8 E. faecium ATCC 29212 Mupirocin 1000.1 MBI 32 11G13CN P. aeruginosa PA41 Ciprofloxacin 4 0.125 MBI 21A1 16P. aeruginosa PA41 Ciprofloxacin 4 1 MBI 21A2 16 P. aeruginosa PA41Ciprofloxacin 8 2 MBI 28 8 P. aeruginosa 001 Piperacillin 128 64 MBI 278 P. aeruginosa 023 Piperacillin 128 64 MBI 29 8 P. aeruginosa 024Tobramycin 64 1 MBI 29 8 P. aeruginosa 025 Ceftazidime 64 16 MBI 29 8 P.aeruginosa 027 Imipenem 16 8 MBI 29 16 P. aeruginosa 028 Imipenem 16 8MBI 29 16 S. haemolyticus SH8578 Erythromycin 8 0.5 MBI 31 1 S. aureusSA7338 Ampicillin 2 0.25 MBI 26 0.25 S. aureus SA7609 Erythromycin 320.5 MBI 26 1 S. aureus SA7835 Erythromycin 8 0.125 MBI 26 2 S. aureusSA7795 Erythromycin 32 1 MBI 26 8 S. aureus SA7796 Erythromycin 32 1 MBI26 2 S. aureus SA7795 Erythromycin 32 4 MBI 31 0.125 S. aureus SA7818Erythromycin 32 2 MBI 31 0.125 S. aureus SA7796 Erythromycin 32 2 MBI 310.125 S. aureus SA7834 Methicillin 32 8 MBI 26 4 S. aureus SA7835Methicillin 32 4 MBI 26 16 S. aureus SA7796 Methicillin 16 2 MBI 31 16S. aureus SA7797 Methicillin 16 2 MBI 31 16 S. aureus SA7823 Methicillin16 2 MBI 31 0.5 S. aureus SA7834 Methicillin 64 1 MBI 31 32 S. aureusSA7835 Methicillin 64 2 MBI 31 16 S. aureus SA007 Piperacillin 128 64MBI 27 0.5 S. aureus MRSA 9 Mupirocin >100 0.1 MBI 2 11D18CN S. aureusMRSA 9 Mupirocin >100 0.1 MBI 8 11G13CN S. aureus MRSA 9 Mupirocin >1000.1 MBI 21A1 16 S. aureus MRSA 9 Mupirocin >100 0.3 MBI 21A10 32 S.aureus MRSA 9 Mupirocin >100 0.1 MBI 21A2 32 S. aureus MRSA 9Mupirocin >100 0.1 MBI 26 4 S. aureus MRSA 9 Mupirocin >100 0.1 MBI 27 2S. aureus MRSA 13 Mupirocin 100 3 MBI 10CN 4 S. aureus MRSA 13 Mupirocin100 0.1 MBI 11CN 16 S. aureus MRSA 13 Mupirocin 100 3 MBI 11F1CN 8 S.aureus 014 Ciprofloxacin 8 0.125 MBI 21A2 4 S. aureus MRSA 17Mupirocin >100 10.3 MBI 10CN 12 S. aureus MRSA 17 Mupirocin >100 1 MBI11A1CN 32 S. aureus MRSA 17 Mupirocin >100 1 MBI 16 11G13CN S. aureusMRSA 17 Mupirocin >100 0.3 MBI 27 2 S. aureus MRSA 17 Mupirocin >100 0.1MBI 29A3 4 S. aureus 093 Ciprofloxacin 32 0.125 MBI 21A1 2 S. aureus 093Ciprofloxacin 32 1 MBI 21A2 4 S. aureus SA 7818 Methicillin 16 4 MBI 262 S. epidermidis SE8497 Clindamycin 32 0.125 MBI 26 2 S. epidermidisSE8403 Erythromycin 8 0.125 MB1 26 2 S. epidermidis SE8410 Erythromycin32 0.5 MBI 26 1 S. epidermidis SE8411 Erythromycin 32 0.5 MBI 26 1 S.epidermidis SE8497 Erythromycin 32 0.125 MBI 26 1 S. epidermidis SE8503Erythromycin 32 0.5 MBI 26 1 S. epidermidis SE8565 Erythromycin 32 0.5MBI 26 1 S. epidermidis SE8403 Erythromycin 8 0.125 MBI 31 2 S.epidermidis SE8410 Erythromycin 32 0.5 MBI 31 1 S. epidermidis SE8411Erythromycin 32 0.5 MBI 31 1 S. epidermidis SE8497 Erythromycin 32 0.125MBI 31 1 S. epidermidis SE8503 Erythromycin 32 0.5 MBI 31 1 S.epidermidis SE8565 Erythromycin 32 0.5 MBI 31 1 S. haemolyticus SH8459Ampicillin 0.5 0.25 MBI 26 0.25 S. haemolyticus SH8472 Ampicillin 2 0.25MBI 26 16 S. haemolyticus SH8564 Ampicillin 64 0.25 MBI 26 32 S.haemolyticus SH8575 Ampicillin 0.5 0.25 MBI 26 8 S. haemolyticus SH8578Ampicillin 0.5 0.25 MBI 26 4 S. haemolyticus SH8597 Clindamycin 16 0.125MBI 26 1 S. haemolyticus SH8463 Erythromycin 8 0.5 MBI 26 0.5 S.haemolyticus SH8472 Erythromycin 8 0.5 MBI 26 0.5 S. haemolyticus SH8575Erythromycin 32 2 MBI 26 0.5 S. haemolyticus SH8578 Erythromycin 8 0.5MBI 26 01 S. haemolyticus SH8597 Erythromycin 32 0.5 MBI 26 0.5 S.haemolyticus SH8463 Erythromycin 8 0.5 MBI 31 0.5 S. haemolyticus SH8472Erythromycin 8 0.5 MBI 31 0.5 S. haemolyticus SH8564 Erythromycin 32 2MBI 31 0.5 S. haemolyticus SH8575 Erythromycin 32 2 MBI 31 0.5 S.haemolyticus SH8563 Methicillin 64 0.25 MBI 26 2 S. maltophilia 034Tobramycin 8 1 MBI 29 4 S. maltophilia 037 Tobramycin 32 4 MBI 29 16 S.maltophilia 039 Ciprofloxacin 4 2 MBI 29 16 S. maltophilia 041Tobramycin 16 1 MBI 29 8 S. maltophilia 043 Imipenem >256 4 MBI 29 16 S.maltophilia 044 Piperacillin >512 16 MBI 26 32

TABLE 24 Teicoplanin (μg/ml) MBI 26 (μg/ml) Microorganism Strain Alone +MBI 26 Alone + Teicoplanin E. faecium 97017 VanA 32 0.25 64 4 E. faecium97018 VanA 32 0.25 64 8 E. faecium 97019 VanA 32 0.5 64 16 E. faecium97020 VanA 32 0.5 64 16 E. faecium 97021 VanA 32 0.5 64 32 E. faecium97022 VanA 32 0.5 64 4 E. faecium 97023 VanA 32 0.25 64 4 E. faecium97024 VanA 32 0.25 64 8 E. faecium 97025 VanA 32 0.5 16 4 E. faecium97026 VanA 32 0.5 64 16 E. faecium 97027 VanA 32 8 64 8 E. faecium 97028VanA 32 0.25 8 8 E. faecium 97029 VanA 32 0.25 64 8 E. faecium 97030VanA 32 0.25 64 32 E. faecium 97031 VanA 32 0.25 64 32 E. faecium 97032VanA 32 0.25 64 8 E. faecium 97033 VanA 32 0.25 64 8 E. faecium 97034VanA 32 0.25 64 8 E. faecium 97035 VanA 32 0.25 64 0.5 E. faecium 97036VanA 8 0.25 8 4 E. faecalis 97050 VanA 32 0.25 64 8 E. faecalis 97051VanA 32 0.25 64 8 E. faecalis 97052 VanA 32 0.25 64 8 E. faecalis 97053VanA 32 0.25 64 8 E. faecalis 97054 VanA 32 0.25 64 8 E. faecalis 97055VanA 32 0.25 64 8

TABLE 25 1. Amikacin Amikacin MIC (μg/ml) Peptide MIC (μg/ml) PeptideOrganism FIC Alone +Peptide Alone +Amikacin MBI 11B16CN P. aeruginosaPA022 0.38 64 8 64 16 MBI 21A2 P. aeruginosa PA022 0.50 64 16 8 2 E.faecium EFM020 0.56 32 2 128 64 E. faecalis EFS008 0.19 64 8 >128 16 MBI26 E. faecium EFM004 0.56 128 8 64 32 E. faecium EFM020 0.75 32 8 64 32MBI 27 E. faecium EFM004 0.75 64 16 16 8 E. faecium EFM020 0.63 32 4 168 E. faecalis EFS008 0.56 32 16 4 0.25 MBI 29A3 E. faecium EFM004 0.56128 8 8 4 E. faecium EFM020 1.00 32 16 4 2 MBI 29F1 E. faecium EFM0040.53 >128 8 32 16 E. faecalis EFS008 0.19 64 4 4 0.5 Deber A2KA2 E.faecalis EFS008 0.19 64 8 >128 16 2. Ceftriaxone Ceftriaxone MIC (μg/ml)Peptide MIC (μg/ml) Peptide Organism FIC Alone +Peptide Alone+Ceftriaxone MBI 11B7CN A. baumannii ABI002 0.50 32 8 32 8 A. baumanniiABI005 0.56 16 8 16 1 MBI 11J02CN A. baumannii ABI005 0.56 16 8 8 0.5 A.lwoffii ALW007 0.75 16 4 4 2 B. cepacia BC003 0.63 16 8 >128 32 E.cloacae ECL014 0.50 128 0.25 32 16 E. cloacae ECL015 0.52 64 1 32 16 MBI26 A. baumannii ABI005 0.53 16 0.5 2 1 A. baumannii ABI006 0.56 128 8 21 B. cepacia BC003 0.50 16 8 >128 0.125 E. cloacae ECL015 0.19 64 4 32 43. Ciprofloxacin Ciprofloxacin MIC (μg/ml) Peptide MIC (μg/ml) PeptideOrganism FIC Alone +Peptide Alone +Ciprofloxacin MBI 11A1CN S. aureusSA10 0.50 32 0.125 128 64 S. aureus SA25 0.53 4 0.125 16 8 MBI 11D18CNP. aeruginosa PA77 0.50 2 0.5 128 32 MBI 21A1 S. aureus SA25 0.16 40.125 32 4 P. aeruginosa PA41 0.50 4 1 16 4 P. aeruginosa PA77 1.00 2 132 16 MBI 21A2 S. aureus SA25 0.56 2 1 16 1 P. aeruginosa PA41 0.50 4 164 16 P. aeruginosa PA77 0.63 2 0.25 64 32 MBI 26 A. calcoaceticus 50.38 2 0.25 >32 16 E. cloacae 16 0.38 2 0.25 >32 16 E. cloacae 17 0.38 20.25 >32 16 P. aeruginosa PA41 0.50 4 1 128 32 P. aeruginosa PA77 0.56 20.125 128 64 P. aeruginosa 30 0.09 4 0.25 >32 2 P. aeruginosa 31 0.27 160.25 >32 16 S. maltophilia 34 0.25 2 0.25 >32 8 S. maltophilia 35 0.50 20.5 >32 16 MBI 27 S. aureus SA25 0.75 4 1 2 1 MBI 28 S. aureus SA25 0.562 0.125 2 1 MBI 29 A. calcoaceticus 3 0.63 8 1 >16 16 A. calcoaceticus 40.63 8 1 >16 16 E. cloacae 16 0.63 2 0.25 >16 16 E. cloacae 17 0.75 2 116 4 S. aureus SA10 0.50 32 0.125 4 2 S. aureus SA14 0.63 8 1 8 4 P.aeruginosa PA41 0.63 8 1 8 4 P. aeruginosa PA77 0.50 2 0.5 64 16 P.aeruginosa 30 0.56 4 0.25 >16 16 P. aeruginosa 31 0.53 16 0.5 >16 16 S.maltophilia 34 0.63 2 0.25 >16 16 S. maltophilia 35 0.63 2 0.25 >16 16MBI 29A2 S. aureus SA10 0.52 32 0.5 4 2 S. aureus SA25 0.63 4 0.5 2 1 P.aeruginosa PA41 1.00 4 2 8 4 P. aeruginosa PA77 1.00 2 1 16 8 MBI 29A3S. aureus SA25 0.75 4 1 1 0.5 P. aeruginosa PA41 0.63 4 0.5 8 4 4.Gentamicin Gentamicin MIC (μg/ml) Peptide MIC (μg/ml) Peptide OrganismFIC Alone +Peptide Alone +Gentamicin MBI 11B16CN A. baumannii ABI0010.31 64 4 16 4 A. baumannii ABI002 0.31 32 2 16 4 A. calcoacetious AC0010.25 8 1 32 4 P. aeruginosa PA023 0.56 8 4 >128 16 P. aeruginosa PA0410.31 8 2 >128 16 S. maltophilia SMA017 0.16 64 2 128 16 S. maltophiliaSMA019 0.51 64 0.5 32 16 MBI 21A2 A. calcoaceticus AC001 1.00 8 4 16 8P. aeruginosa PA022 0.56 32 2 8 4 S. maltophilia SMA020 0.50 64 0.125 168 S. maltophilia SMA021 0.50 64 0.125 16 8 MBI 26 A. baumannii ABI0010.56 64 4 8 4 A. baumannii ABI002 0.53 16 0.5 8 4 P. aeruginosa PA0230.75 8 4 >128 64 P. aeruginosa PA041 0.75 8 4 64 16 S. maltophiliaSMA017 0.52 64 1 16 8 S. maltophilia SMA019 0.53 64 2 4 2 MBI 27 A.baumannii ABI002 0.52 32 0.5 8 4 A. calcoaceticus AC001 0.63 8 1 8 4 P.aeruginosa PA023 0.50 16 4 32 8 P. aeruginosa PA041 1.00 8 4 16 8 S.maltophilia SMA019 0.50 64 0.125 8 4 S. maltophilia SMA020 0.50 64 0.1258 4 MBI 29A3 A. baumannii ABI002 0.75 16 4 2 1 P. aeruginosa PA041 1.008 4 8 4 MBI 29F1 A. calcoaceticus AC001 0.75 8 2 8 4 P. aeruginosa PA0230.52 8 0.125 128 64 Debar A2KA2 A. calcoaceticus AC001 0.56 8 4 >128 16P. aeruginosa PA041 0.50 16 4 >128 64 5. Mupirocin Mupirocin MIC (μg/ml)Peptide MIC (μg/ml) Peptide Organism FIC Alone +Peptide Alone +MupirocinMBI 27 S. aureus SBSA4 0.50 >100 0.3 4 2 6. Piperacillin PiperacillinMIC (μg/ml) Peptide MIC (μg/ml) Peptide Organism FIC Alone +PeptideAlone +Piperacillin MBI 11B7CN S. maltophilia 2 1.00 32 16 128 8 S.marcescens 1 0.27 32 8 >128 4 H. influenzae 1 0.13 64 8 >128 1 MBI11B9CN A. calcoaceticus 3 0.75 64 16 32 16 S. maltophilia 2 0.75 64 1632 16 S. marcescens SB1 0.26 64 16 >128 2 P. aeruginosa 12 0.75 >128 64128 64 P. aeruginosa 15 0.50 >128 64 >128 64 MBI 11CN A. calcoaceticus 31.00 32 16 64 32 S. maltophilia 2 0.75 64 16 64 32 P. aeruginosa 220.52 >128 4 64 32 P. aeruginosa 23 0.53 128 64 128 4 MBI 11 D18CN A.calcoaceticus 3 0.38 64 8 32 8 E. cloacae 9 0.31 >128 16 64 16 E.cloacae 10 0.56 >128 16 32 16 S. maltophilia 2 0.50 64 16 32 8 S.maltophilia 14 0.63 128 16 16 8 S. marcescens 1 0.14 64 8 >128 4 P.aeruginosa 23 0.56 128 64 64 4 MBI 11E3CN A. calcoaceticus 3 0.75 32 1632 8 S. maltophilia 3 0.75 64 16 32 16 S. maltophilia 4 0.75 64 16 32 16S. marcescens SB1 0.26 64 16 >128 2 P. aeruginosa 7 1.00 128 64 64 32 P.aeruginosa 23 0.27 128 32 64 1 H. influenzae 1 0.38 64 8 >128 64 H.influenzae 2 0.31 32 8 >128 16 MBI 11F3CN A. calcoaceticus 3 0.63 32 1632 4 S. maltophilia 2 0.75 64 16 32 16 P. aeruginosa 7 1.00 128 64 12864 P. aeruginosa 23 0.51 128 64 64 0.5 MBI 11F4CN E. cloacae 100.52 >128 4 16 8 S. maltophilia 2 0.50 64 16 16 4 S. marcescens 10.08 >128 16 >128 4 P. aeruginosa 7 0.38 >128 64 64 8 P. aeruginosa 230.31 >128 64 64 4 H. influenzae 1 0.75 32 16 >128 64 MBI 11G7CN A.calcoaceticus 3 0.63 128 16 64 32 S. maltophilia 2 0.75 64 16 64 16 S.marcescens 1 0.25 64 16 >128 1 P. aeruginosa 7 0.50 >128 64 >128 64 P.aeruginosa 23 0.50 128 64 >128 1 H. influenzae 1 0.75 32 16 >128 64 MBI21A2 E. coli 1 0.53 >128 8 4 2 S. maltophilia 3 0.75 64 16 32 16 S.maltophilia 11 0.75 32 8 128 64 S. marcescens 1 0.27 64 16 >128 4 H.influenzae 1 0.31 64 4 >128 64 H. influenzae 2 0.28 128 4 >128 64 MBI 26S. maltophilla 2 0.75 64 16 4 2 S. maltophilia 4 0.63 128 16 16 8 S.marcescens 1 0.09 64 2 >128 16 P. aeruginosa 7 0.25 >128 32 >128 32 H.influenzae 1 0.19 64 4 >128 32 H. influenzae 2 0.19 128 16 >128 16 A.calcoaceticus 2 0.50 >512 4 32 16 A. calcoaceticus 7 0.25 32 4 >32 8 E.cloacae 13 0.16 128 4 >32 8 E. cloacae 19 0.31 64 4 >32 16 P. aeruginosa23 0.27 256 4 >64 32 P. aeruginosa 26 0.56 128 8 >32 32 S. maltophilia35 0.26 >256 4 >32 16 S. maltophilia 41 0.52 >512 16 >32 32 MBI 29 S.marcescens 1 0.09 64 16 >128 8 P. aeruginosa 23 0.63 128 64 16 2 H.influenzae 1 0.51 32 16 16 0.125 A. calcoaceticus 2 0.50 >512 4 16 8 A.calcoaceticus 7 0.25 32 4 >16 4 E. cloacae 16 0.50 >512 4 >16 16 E.cloacae 17 0.50 >512 4 >16 16 P. aeruginosa 23 0.63 128 64 >32 8 P.aeruginosa 24 0.50 >512 4 >16 16 S. maltophilia 34 0.25 >512 4 >16 8 S.maltophilia 35 0.50 >512 4 >16 16 7. Tobramycin Tobramycin MIC (μg/ml)Peptide MIC (μg/ml) Peptide Organism FIC Alone +Peptide Alone+Tobramycin MBI 11A1CN P. aeruginosa PA026 0.50 8 4 >128 0.125 S.maltophilia SMA029 0.16 128 4 >128 32 S. maltophilia SMA030 0.27 1282 >128 64 MBI 11B9CN A. baumannii ABI001 0.50 16 4 32 8 E. coli ECO0060.75 8 4 32 8 P. aeruginosa PA008 0.50 32 0.125 128 64 P. aeruginosaPA025 0.56 32 2 128 64 S. maltophilia SMA027 0.63 8 4 >128 32 S.maltophilia SMA031 0.19 64 4 >128 32 MBI 11CN A. baumannii ABI001 0.5016 4 64 16 E. coli ECO006 0.53 8 4 8 0.25 P. aeruginosa PA032 0.50 164 >128 64 S. maltophilia SMA029 0.27 128 2 >128 64 S. maltophilia SMA0300.27 128 2 >128 64 MBI 11D18CN A. baumannii ABI001 0.31 16 4 64 4 A.baumannii ABI002 0.53 8 4 16 0.5 P. aeruginosa PA032 1.00 8 4 64 32 S.maltophilia SMA027 0.19 32 4 >128 16 S. maltophilia SMA029 0.27 128 2 328 S. epidermidis SE080 0.75 16 4 2 1 MBI 11F3CN A. baumannii ABI001 0.5316 0.5 32 16 P. aeruginosa PA032 0.50 16 4 >128 64 S. maltophilia SMA0290.26 128 1 128 32 S. maltophilia SMA030 0.26 128 1 128 32 MBI 11G13CN A.baumannii ABI001 0.50 16 4 128 32 P. aeruginosa PA022 0.56 8 4 >128 16MBI 21A1 P. aeruginosa PA022 0.53 8 4 4 0.125 P. aeruginosa PA026 0.51 84 16 0.125 P. aeruginosa PA030 0.52 16 0.25 16 8 P. aeruginosa PA0320.63 8 1 64 32 S. maltophilia SMA029 0.28 128 4 128 32 S. maltophiliaSMA030 0.16 128 4 >128 32 MBI 22A1 A. baumannii ABI001 0.75 16 4 4 2 S.maltophilia SMA029 0.51 128 1 16 8 S. maltophilia SMA029 0.50 128 0.12532 16 S. epidermidis SE072 0.50 >128 0.125 16 8 S. epidermidis SE0730.50 >128 0.125 16 8 MBI 26 P. aeruginosa PA031 0.75 16 4 32 16 S.maltophilia SMA027 0.50 16 4 >128 64 S. epidermidis SE068 0.27 >128 4 20.5 S. epidermidis SE071 0.50 >128 0.125 16 8 MBI 27 E. coli ECO006 0.568 0.5 8 4 S. maltophilia SMA027 1.00 8 4 32 16 S. maltophilia SMA0310.53 128 4 16 8 MBI 29 E. coli ECO006 0.53 8 4 4 0.125 P. aeruginosaPA032 1.00 8 4 128 64 S. maltophilia SMA031 0.50 >128 0.25 16 8 S.maltophilia SMA032 0.53 128 4 16 8 MBI 29A3 E. coli ECO006 0.75 8 2 4 2P. aeruginosa PA022 0.56 8 4 4 0.25 S. maltophilia SMA027 0.75 16 4 3216 S. maltophilia SMA029 0.28 128 4 16 4 REWH S. maltophilia SMA029 0.13128 0.25 >128 32 53A5CN S. maltophilia SMA030 0.13 128 0.25 >128 32 8.Vancomycin Vancomycin MIC (μg/ml) Peptide MIC (μg/ml) Peptide OrganismFIC Alone +Peptide Alone +Vancomycin MBI 11A1CN E. faecalis EFS003 0.638 4 >128 32 E. faecalis EFS006 0.50 8 4 128 0.25 E. faecalis EFS010 0.1316 1 128 8 E. faecalis EFS014 0.51 128 1 4 2 E. faecium EFM004 0.50 >1280.5 32 16 E. faecium EFM007 0.28 128 4 64 16 E. faecium EFM009 0.25 32 464 8 MBI 11D18CN E. faecalis EFS003 0.75 8 2 64 32 E. faecalis EFS0070.63 8 1 16 8 E. faecalis EFS009 0.75 8 2 8 4 E. faecium EFM0040.50 >128 0.5 8 4 E. faecium EFM007 0.50 >128 0.5 8 4 E. faecium EFM0090.52 64 1 8 4 E. faecium EFM010 0.50 >128 1 8 4 MBI 21A1 E. faecalisEFS012 0.09 128 4 32 2 E. faecalis EFS013 0.09 128 4 32 2 E. faeciumEFM010 0.56 64 4 32 16 MBI 26 E. faecalis EFS005 0.31 16 4 >128 16 E.faecalis EFS010 0.27 64 1 4 1 E. faecalis EFS011 0.25 16 2 >128 32 E.faecium EFM004 0.25 >128 0.125 64 16 E. faecium EFM010 0.53 128 1 32 16E. faecium EFM011 0.31 32 2 32 8 MBI 29 E. faecalis EFS012 0.50 >128 1 21 E. faecalis EFS013 0.50 >128 1 2 1 E. faecium EFM005 0.53 128 4 8 4 E.faecium EFM009 0.75 16 4 8 4 E. faecium EFM010 0.63 32 4 8 4 E. faeciumEFM016 0.51 128 1 4 2 MBI 29A3 E. faecalis EFS005 0.19 16 1 32 4 E.faecalis EFS011 0.50 16 4 32 8 E. faecalis EFS014 0.52 64 1 1 0.5 E.faecium EFM006 0.52 >128 4 4 2

These data show that acquired resistance can be overcome. For example,the acquired resistance of S. aureus, a Gram-positive organism, topiperacillin and ciprofloxacin is overcome when these antibiotic agentsare combined with peptides MBI 27, MBI 21A1 or MBI 21A2 respectively.Similar results are obtained for peptides MBI 26 and MBI 31 incombination with methicillin, ampicillin and erythromycin, and forpeptide MBI 26 in combination with vancomycin or teicoplanin againstresistant enterococci.

Example 9 Synergy of Cationic Peptides and Lysozyme or Nisin

The effectiveness of the antibiotic activity of lysozyme or nisin isimproved when either agent is administered in combination with acationic peptide. The improvement is demonstrated by measurement of theMICs of lysozyme or nisin alone and in combination with the peptide,whereby the lysozyme or nisin MIC is lower in combination than alone.The MICs can be measured by the agarose dilution assay, the brothdilution assay or by time kill curves.

Example 10 Biochemical Characterization of Peptide Analogues Solubilityin Formulation Buffer

The primary factor affecting solubility of a peptide is its amino acidsequence. Polycationic peptides are preferably freely soluble in aqueoussolutions, especially under low pH conditions. However, in certainformulations, polycationic peptides may form an aggregate that isremoved in a filtration step. As peptide solutions for in vivo assaysare filtered prior to administration, the accuracy and reproducibilityof dosing levels following filtration are examined.

Peptides dissolved in formulations are filtered through a hydrophilic0.2 μm filter membrane and then analyzed for total peptide content usingreversed-phase HPLC. A 100% soluble standard for each concentration isprepared by dissolving the peptide in MilliQ water. Total peak area foreach condition is measured and compared with the peak area of thestandard in order to provide a relative recovery value for eachconcentration/formulation combination.

MBI 11CN was prepared in four different buffer systems (A, B, C, and C1)(Table 26, below) at 50, 100, 200 and 400 μg/ml peptide concentrations.With formulations A or B, both commonly used for solvation of peptidesand proteins, peptide was lost through filtration in a concentrationdependent manner (FIG. 4). Recovery only reached a maximum of 70% at aconcentration of 400 μg/ml. In contrast, peptides dissolved informulations C and C1 were fully recovered. Buffers containingpolyanionic ions appear to encourage aggregation, and it is likely thatthe aggregate takes the form of a matrix which is trapped by the filter.Monoanionic counterions are more suitable for the maintenance ofpeptides in a non-aggregated, soluble form, while the addition of othersolubilizing agents may further improve the formulation.

TABLE 26 Code Formulation Buffer A PBS 200 mM, pH 7.1 B Sodium Citrate100 mM, pH 5.2 C Sodium Acetate 200 mM, pH 4.6 C1 Sodium Acetate 200mM/0.5% Polysorbate 80, pH 4.6 D Sodium Acetate 100 mM/0.5% ActivatedPolysorbate 80, pH 7.5: Lyophilized/Reconstituted

Solubility in Broth

The solubility of peptide analogues is assessed in calcium and magnesiumsupplemented Mueller Hinton broth by visual inspection. The procedureemployed is that used for the broth dilution assay except that bacteriaare not added to the wells. The appearance of the solution in each wellis evaluated according to the scale: (a) clear, no precipitate, (b)light diffuse precipitate and (c) cloudy, heavy precipitate. Resultsshow that, for example, MBI 10CN is less soluble than MBI 11CN underthese conditions and that MBI 11BCN analogues are less soluble than MBI11ACN analogues.

Reversed Phase HPLC Analysis of Peptide Analogue Formulations

Reversed-phase HPLC, which provides an analytical method for peptidequantification, is used to examine peptides in two differentformulations. A 400 μg/mL solution of MBI 11CN prepared in formulationsC1 and D is analyzed by using a stepwise gradient to resolve freepeptide from other species. Standard chromatographic conditions are usedas follows:

-   -   Solvent A: 0.1% trifluoroacetic acid (TFA) in water    -   Solvent B: 0.1% TFA/95% acetonitrile in water    -   Media: POROS® R2-20 (polystyrene divinylbenzene)

As shown in FIG. 5, MBI 11CN could be separated in two forms, as freepeptide in formulation C1, and as a principally formulation-complexpeptide in formulation D. This complex survives the separation protocolin gradients containing acetonitrile, which might be expected to disruptthe stability of the complex. A peak corresponding to a small amount(<10%) of free peptide is also observed in formulation D. If the shapeof the elution gradient is changed, the associated peptide elutes as abroad low peak, indicating that complexes of peptide in the formulationare heterogeneous.

Example 11 Structural Analysis of Indolicidin Variants Using CircularDichroism Spectroscopy

Circular dichroism (CD) is a spectroscopic technique that measuressecondary structures of peptides and proteins in solution, see forexample, R. W. Woody, (Methods in Enzymology, 246: 34, 1995). The CDspectra of α-helical peptides is most readily interpretable due to thecharacteristic double minima at 208 and 222 nm. For peptides with othersecondary structures however, interpretation of CD spectra is morecomplicated and less reliable. The CD data for peptides is used torelate solution structure to in vitro activity.

CD measurements of indolicidin analogues are performed in threedifferent aqueous environments, (1) 10 mM sodium phosphate buffer, pH7.2, (2) phosphate buffer and 40% (v/v) trifluoroethanol (TFE) and (3)phosphate buffer and large (100 nm diameter) unilamellar phospholipidvesicles (liposomes) (Table 27). The organic solvent TFE and theliposomes provide a hydrophobic environment intended to mimic thebacterial membrane where the peptides are presumed to adopt an activeconformation.

The results indicate that the peptides are primarily unordered inphosphate buffer (a negative minima at around 200 nm) with the exceptionof MBI 11F4CN, which displays an additional minima at 220 nm (seebelow). The presence of TFE induces p-turn structure in MBI 11 and MBI11G4CN, and increases α-helicity in MBI 11F4CN, although most of thepeptides remain unordered. In the presence of liposomes, peptides MBI11CN and MBI 11B7CN, which are unordered in TFE, display p-turnstructure (a negative minima at around 230 nm) (FIG. 6). Hence,liposomes appear to induce more ordered secondary structure than TFE.

A β-turn is the predominant secondary structure that appears in ahydrophobic environment, suggesting that it is the primary conformationin the active, membrane-associated form. In contrast, MBI 11F4CNdisplays increased α-helical conformation in the presence of TFE.Peptide MBI 11F4CN is also the most insoluble and hemolytic of thepeptides tested, suggesting that α-helical secondary structure mayintroduce unwanted properties in these analogues.

Additionally CD spectra are recorded for APO-modified peptides (Table28). The results show that these compounds have significant p-turnsecondary structure in phosphate buffer, which is only slightly alteredin TFE.

Again, the CD results suggest that a β-turn structure (i.e.membrane-associated) is the preferred active conformation among theindolicidin analogues tested.

TABLE 27 Phosphate buffer Conformation TFE Conformation Peptide min λmax λ in buffer min λ max λ in TFE MBI 10CN 201 — Unordered 203 ~219  Unordered MBI 11 199 — Unordered 202, 227 220 β-turn MBI 11ACN 199 —Unordered 203 219 Unordered MBI 11CN 200 — Unordered 200 — Unordered MBI11CNY1 200 — Unordered 200 — Unordered MBI 11B1CNW1 201 — Unordered 201— Unordered MBI 11B4ACN 200 — Unordered 200 — Unordered MBI 11B7CN 200 —Unordered   204, ~219 Unordered MBI 11B9ACN 200 — Unordered 200 —Unordered MBI 11B9CN 200 — Unordered 200 — Unordered MBI 11D1CN 200 —Unordered 204 — Unordered MBI 11E1CN 201 — Unordered 201 — Unordered MBI11E2CN 200 — Unordered 201 — Unordered MBI 11E3CN 202 226 ppll helix 200— Unordered MBI 11F3CN 199 228 ppll helix 202 — Unordered MBI 11F4CN202, 220 — Unordered 206, 222 — slight α-helix MBI 11G4CN 199, 221 —Unordered 201, 226 215 β-turn MBI 11G6ACN 200 — Unordered 199 —Unordered MBI 11G7ACN 200 — Unordered 202 221 Unordered

TABLE 28 Phosphate Confor- Confor- APO-modified buffer mation TFE mationpeptide min λ max λ in buffer min λ max λ in TFE MBI 11CN 202, 220β-turn 203 223 β-turn 229 MBI 11BCN 200, — β-turn 202 222 β-turn 229 MBI11B7CN 202, 223 β-turn 199 230 β-turn 230 MBI 11E3CN 202, 220 β-turn 199— β-turn 229 MBI 11F3CN 205 — ppll helix 203 230 ppll helix

Example 12 Membrane Permeabilization Assays Liposome Dye Release

A method for measuring the ability of peptides to permeabilizephospholipid bilayers is described (Parente et al., Biochemistry, 29,8720, 1990) Briefly, liposomes of a defined phospholipid composition areprepared in the presence of a fluorescent dye molecule. In this example,a dye pair consisting of the fluorescent molecule8-aminonapthalene-1,3,6-trisulfonic acid (ANTS) and its quenchermolecule p-xylene-bis-pyridinium bromide (DPX) are used. The mixture offree dye molecules, dye free liposomes, and liposomes containingencapsulated ANTS-DPX are separated by size exclusion chromatography. Inthe assay, the test peptide is incubated with the ANTS-DPX containingliposomes and the fluorescence due to ANTS release to the outside of theliposome is measured over time.

Using this assay, peptide activity, measured by dye release, is shown tobe extremely sensitive to the composition of the liposomes at manyliposome to peptide ratios (L/P) (FIG. 7). Specifically, addition ofcholesterol to liposomes composed of egg phosphotidylcholine (PC)virtually abolishes membrane permeabilizing activity of MBI 11CN, evenat very high lipid to peptide molar ratios (compare with egg PCliposomes containing no cholesterol). This in vitro selectivity maymimic that observed in vitro for bacterial cells in the presence ofmammalian cells.

In addition, there is a size limitation to the membrane disruptioninduced by MBI 11CN. ANTS/DPX can be replaced with fluoresceinisothiocyanate-labeled dextran (FD-4), molecular weight 4,400, in theegg PC liposomes. No increase in FD-4 fluorescence is detected uponincubation with MBI 11CN. These results indicate that MBI 11CN-mediatedmembrane disruption allows the release of the relatively smallerANTS/DPX molecules (˜400 Da), but not the bulkier FD-4 molecules.

E. coli ML-35 Inner Membrane Assay

An alternative method for measuring peptide-membrane interaction usesthe E. coli strain ML-35 (Lehrer et al., J. Clin. Invest., 84: 553,1989), which contains a chromosomal copy of the lacZ gene encodingβ-galactosidase and is permease deficient. This strain is used tomeasure the effect of peptide on the inner membrane through release ofβ-galactosidase into the periplasm. Release of β-galactosidase ismeasured by spectrophotometrically monitoring the hydrolysis of itssubstrate o-nitrophenol β-D-galactopyranoside (ONPG). The maximum rateof hydrolysis (V_(max)) is determined for aliquots of cells taken atvarious growth points.

A preliminary experiment to determine the concentration of peptiderequired for maximal activity against mid-log cells, diluted to 4×10⁷CFU/ml, yields a value of 50 μg/ml, which is used in all subsequentexperiments. Cells are grown in two different growth media, Terrificbroth (TB) and Luria broth (LB) and equivalent amounts of cells areassayed during their growth cycles. The resulting activity profile ofMBI 11B7CN is shown in FIG. 8. For cells grown in the enriched TB media,maximum activity occurs at early mid-log (140 min), whereas for cellsgrown in LB media, the maximum occurs at late mid-log (230 min).Additionally, only in LB, a dip in activity is observed at 140 min. Thisdrop in activity may be related to a transition in metabolism, such as arequirement for utilization of a new energy source due to depletion ofthe original source, which does not occur in the more enriched TB media.A consequence of a metabolism switch would be changes in the membranepotential.

To test whether membrane potential has an effect on peptide activity,the effect of disrupting the electrochemical gradient using thepotassium ionophore valinomycin is examined. Cells pre-incubated withvalinomycin are treated with peptide and for MBI 10CN and MBI 11CN ONPGhydrolysis diminished by approximately 50% compared to no pre-incubationwith valinomycin (FIG. 9). Another cationic peptide that is notsensitive to valinomycin is used as a positive control.

Further delineation of the factors influencing membrane permeabilizingactivity are tested. In an exemplary test, MBI 11B7CN is pre-incubatedwith isotonic HEPES/sucrose buffer containing either 150 mM sodiumchloride (NaCl) or 5 mM magnesium ions (Mg²⁺) and assayed as describedearlier. In FIG. 10, a significant inhibition is observed with eithersolution, suggesting involvement of electrostatic interactions in thepermeabilizing action of peptides.

Example 13 Erythrocyte Hemolysis by Cationic Peptides

Cationic peptides are tested for toxicity towards eukaryotic cells bymeasuring the extent of lysis of mammalian red blood cells (RBC).Briefly, in this assay, red blood cells are separated from whole bloodby centrifugation and washed free of plasma components. A 5% (v/v)washed red blood cell suspension is prepared in isotonic saline. Analiquot of peptide in formulation is then added and mixed in. Afterincubation at 37° C. for 1 hour with constant agitation, the solution iscentrifuged and the supernatant measured for absorbance at 540 nm todetect released hemoglobin. When compared with the absorbance for a 100%lysed standard, a relative measure of the amount of hemoglobin that hasbeen released from inside the red blood cells is determined and hencethe ability of the peptide/formulation to cause red blood cell lysis.

Three peptide analogues, MBI 10CN, MBI 11 and MBI 11CN, in formulationC1 at 800 μg/ml cause substantial lysis, which is due primarily to thepH of the buffer. In contrast, formulation D has a more neutral pH andcauses significantly less lysis. Under these conditions, MBI 10CN, MBI11, and MBI 11CN are essentially non-lytic, resulting in 3.9, 2.3, and3.2% lysis, respectively.

Various cationic peptides are tested for the extent of erythrocytelysis. As shown in the following table, very little toxicity isobserved.

TABLE 29 Peptide # % Lysis Apidaecin IA 0.3 MBI 10CN 4.3 MBI 11CN 0.8MBI 11A1CN 0.5 MBI 11A2C N 0.1 MBI 11A3CN 0.0 MBI 11A4CN 0.3 MBI 11A5CN0.3 MBI 11A6CN 0.7 MBI 11A7CN 0.5 MBI 11B1CN 3.1 MBI 11B2CN 3.2 MBI11B3CN 3.3 MBI 11B4CN 1.6 MBI 11B5CN 1.7 MBI 11B7CN 3.2 MBI 11B8CN 1.1MBI 11B9CN 0.4 MBI 11B10CN 0.2 MBI 11D3CN 0.8 MBI 11D4CN 0.9 MBI 11D5CN0.7 MBI 11D6CN 1.1 MBI 11D11H 0.7 MBI 11D13H 1.7 MBI 11D14CN 1.1 MBI11D15CN 0.9 MBI 11D18CN 0.8 MBI 11E1CN 0.8 MBI F11E2CN 0.5 MBI 11E3CN1.3 MBI 11F1CN 2.1 MBI 11F2CN 1.4 MBI 11G3CN 0.5 MBI 11G5CN 0.6 MBI11G6CN 0.6 MBI 11G7CN 1.5 MBI 11G13CN 0.2 MBI 11G14CN 1.1 MBI 21A2 0.5MBI 26 0.6 MBI 27 2.7 MBI 28 4.7 MBI 29 1.9 MBI 29A3 2.0 MBI 31 0.3

A combination of cationic peptide and antibiotic agent is tested fortoxicity towards eukaryotic cells by measuring the extent of lysis ofmammalian red blood cells. Briefly, red blood cells are separated fromwhole blood by centrifugation, washed free of plasma components, andresuspended to a 5% (v/v) suspension in isotonic saline. The peptide andantibiotic agent are pre-mixed in isotonic saline, or other acceptablesolution, and an aliquot of this solution is added to the red blood cellsuspension. Following incubation with constant agitation at 37° C. for 1hour, the solution is centrifuged, and the absorbance of the supernatantis measured at 540 nm, which detects released hemoglobin. Comparison tothe A₅₄₀ for a 100% lysed standard provides a relative measure ofhemoglobin release from red blood cells, indicating the lytic ability ofthe cationic peptide and antibiotic agent combination.

A red blood cell (RBC) lysis assay is used to group peptides accordingto their ability to lyse RBC under standardized conditions compared withMBI 11CN and Gramicidin-S. Peptide samples and washed sheep RBC areprepared in isotonic saline with the final pH adjusted to between 6 and7. Peptide samples and RBC suspension are mixed together to yieldsolutions that are 1% (v/v) RBC and 5, 50 or 500 μg/ml peptide. Theassay is performed as described above. Each set of assays also includesMBI 11CN (500 μg/ml) and Gramicidin-S (5 μg/ml) as “low lysis” and “highlysis” controls, respectively.

MBI11B7CN, MBI11F3CN and MBI11F4CN are tested using this procedure andthe results are presented in Table 30 below.

TABLE 30 % lysis at % lysis at % lysis at Peptide 5 μg/ml 50 μg/ml 500μg/ml MBI 11B7CN 4 13 46 MBI 11F3CN 1 6 17 MBI 11F4CN 4 32 38 MBI 11CNN/D N/D 9 Gramicidin-S 30  N/D N/D N/D = not done

Peptides that at 5 μg/ml lyse RBC to an equal or greater extent thanGramicidin-S, the “high lysis” control, are considered to be highlylytic. Peptides that at 500 μg/ml lyse RBC to an equal to or lesserextent than MBI 11CN, the “low lysis” control, are considered to benon-lytic. The three analogues tested are all “moderately lytic” as theycause more lysis than MBI 11CN and less than Gramicidin S. In additionone of the analogues, MBI-11F3CN, is significantly less lytic than theother two variants at all three concentrations tested.

Example 14 Production of Antibodies to Peptide Analogues

Multiple antigenic peptides (MAPs), which contain four or eight copiesof the target peptide linked to a small non-immunogenic peptidyl core,are prepared as immunogens. Alternatively, the target peptide isconjugated to bovine serum albumin (BSA) or ovalbumin. For example, MBI11B7 conjugated to ovalbumin is used as an immunogen. The immunogens areinjected subcutaneously into rabbits to raise IgG antibodies usingstandard protocols (see, Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1988). After repeated boosters (usually monthly), serum from a bloodsample is tested in an ELISA against the target peptide. A positiveresult indicates the presence of antibodies and further tests determinethe specificity of the antibody binding to the target peptide. Purifiedantibodies can then be isolated from this serum and used in ELISAs toselectively identify and measure the amount of the target peptide inresearch and clinical samples.

Example 15 Pharmacology of Cationic Peptides in Plasma and Blood

The in vitro lifetime of free peptides in plasma and in blood isdetermined by measuring the amount of peptide present after setincubation times. Blood is collected from sheep, treated with ananticoagulant (not heparin) and, for plasma preparation, centrifuged toremove cells. Formulated peptide is added to either the plasma fractionor to whole blood and incubated. Following incubation, peptide isidentified and quantified directly by reversed phase HPLC or anantibody-based assay. The antibiotic agent is quantified by a suitableassay, selected on the basis of its structure. Chromatographicconditions are as described above. Extraction is not required as thefree peptide peak does not overlie any peaks from blood or plasma.

A 1 mg/mL solution of MBI 11CN in formulations C1 and D is added tofreshly prepared sheep plasma at a final peptide concentration of 100μg/mL and incubated at 37° C. At various times, aliquots of plasma areremoved and analyzed for free peptide by reversed phase HPLC. From eachchromatogram, the area of the peak corresponding to free peptide isintegrated and plotted against time of incubation. As shown in FIG. 11,peptide levels diminish over time. Moreover, when administered informulation D, up to 50% of the peptide is immediately released fromformulation-peptide complex on addition to the blood. The decay curvefor free peptide yields an apparent half-life in blood of 90 minutes forboth formulation C1 and D. These results indicate that in sheep's bloodMBI 11CN is relatively resistant to plasma peptidases and proteases. Newpeaks that appeared during incubation may be breakdown products of thepeptide.

A 1 mg/mL solution of MBI 11B7CN in isotonic saline is added to freshlyprepared heat-inactivated rabbit serum, to give a final peptideconcentration of 100 μg/mL and is incubated at 32° C. The peptide levelsdetected are shown in FIG. 12.

A series of peptide stability studies are performed to investigate theaction of protease inhibitors on peptide degradation. Peptide is addedto rabbit serum or plasma, either with or without protease inhibitors,then incubated at 22° C. for 3 hrs. Protease inhibitors tested includeamastatin, bestatin, COMPLETE protease inhibitor cocktail, leupeptin,pepstatin A and EDTA. Amastatin and bestatin at 100 μM prevent thedegradation of MBI 11B7CN in plasma over 3 hrs (FIG. 13). For thisexperiments 10 mM stock solutions of amastatin and bestatin are preparedin dimethylsulfoxide. These solutions are diluted 1:100 inheat-inactivated rabbit serum and incubated at 22° C. for 15 mins priorto addition of peptide. MBI 11B7CN is added to the serum at a finalconcentration of 100 μg/mL and incubated for 3 hrs at 22° C. After theincubation period, the serum samples are analyzed on an analytical C₈column (Waters Nova Pak C₈ 3.9×170 mm) with detection at 280 nm. In FIG.13, MBI 11B7CN elutes at 25 min.

Peptide is extracted from plasma using C₈ Sep Pak cartridges at peptideconcentrations between 0 and 50 μg/mL. Each extraction also contains MBI11CN at 10 μg/mL as an internal standard. Immediately after addition ofthe peptides to fresh rabbit plasma, the samples are mixed then diluted1:10 with a 1% aqueous trifluoroacetic acid (TEA) solution, to give afinal TFA concentration of 0.1%. Five hundred μL of this solution isimmediately loaded onto a C₈ Sep Pak cartridge and eluted with 0.1% TFAin 40% acetonitrile/60% H₂O. Twenty μL of this eluant is loaded onto a4.6×45 mm analytical C₁₈ column and is eluted with an acetonitrilegradient of 25% to 65% over 8 column volumes. The peptides are detectedat 280 nm. A chromatogram showing the extraction MBI 11B7CN with MBI11CN as an internal standard is shown in FIG. 14. MBI 11B7CN and MBI11CN elute at 5 and 3 min respectively. MBI 11B7CN is detected overbackground at concentrations of 5 μg/mL and above.

Peptide levels in plasma in vivo are measured after iv or ipadministration of 80-100% of the maximum tolerated dose of peptideanalogue in either formulation C1 or D. MBI 11CN in formulation C1 isinjected intravenously into the tail vein of CD1 ICRBR strain mice. Atvarious times post-injection, mice are anesthetized and blood is drawnby cardiac puncture. Blood from individual mice is centrifuged toseparate plasma from cells. Plasma is then analyzed by reversed phaseHPLC column. The resulting elution profiles are analyzed for freepeptide content by UV absorbance at 280 nm, and these data are convertedto concentrations in blood based upon a calibrated standard. Each datapoint represents the average blood level from two mice. In this assay,the detection limit is approximately 1 μg/ml, less than 3% of the doseadministered.

The earliest time point at which peptide can be measured is threeminutes following injection, thus, the maximum observed concentration(in μg/ml) is extrapolated back to time zero (FIG. 15). The projectedinitial concentration corresponds well to the expected concentration ofbetween 35 and 45 μg/ml. Decay is rapid, however, and when the curve isfitted to the equation for exponential decay, free circulating peptideis calculated to have a half life of 2.1 minutes. Free circulatingpeptide was not detectable in the blood of mice that were injected withMBI 11CN in formulation D, suggesting that peptide is not released asquickly from the complex as in vitro.

In addition, MBI 11CN is also administered to CD1 ICRBR strain mice by asingle ip injection at an efficacious dose level of 40 mg/kg. Peptide isadministered in both formulations C1 and D to determine if peptidecomplexation has any effect on blood levels. At various times postinjection, mice are anesthetized and blood is drawn by cardiac puncture.Blood is collected and analyzed as for the iv injection.

MBI 11CN administered by this route demonstrated a quite differentpharmacologic profile (FIG. 16). In formulation C1, peptide entered theblood stream quickly, with a peak concentration of nearly 5 μg/ml after15 minutes, which declined to non-detectable levels after 60 minutes. Incontrast, peptide in formulation D is present at a level above 2 μg/mlfor approximately two hours. Therefore, formulation affects entry into,and maintenance of levels of peptide in the blood.

The in vivo lifetime of the cationic peptide and antibiotic agentcombination is determined by administration, typically by intravenous orintraperitoneal injection, of 80-100% of the maximum tolerable dose ofthe combination in a suitable animal model, typically a mouse. At settimes post-injection, each group of animals are anesthetized, blood isdrawn, and plasma obtained by centrifugation. The amount of peptide oragent in the plasma supernatant is analyzed as for the in vitrodetermination.

Example 16 Toxicity of Cationic Peptides In Vivo

The acute, single dose toxicity of various indolicidin analogues istested in Swiss CD1 mice using various routes of administration. Inorder to determine the inherent toxicities of the peptide analogues inthe absence of any formulation/delivery vehicle effects, the peptidesare all administered in isotonic saline with the final pH between 6 and7.

Intraperitoneal route. Groups of 6 mice are injected with peptide dosesof between 80 and 5 mg/kg in 500 μl dose volumes. After peptideadministration, the mice are observed for a period of 5 days, at whichtime the dose causing 50% mortality (LD₅₀), the dose causing 90-100%mortality (LD₉₀₋₁₀₀) and maximum tolerated dose (MTD) levels aredetermined. The LD₅₀ values are calculated using the method of Reed andMuench (J. of Amer. Hyg. 27: 493-497, 1938). The results presented inTable 31 show that the LD₅₀ values for MBI 11CN and analogues range from21 to 52 mg/kg.

TABLE 31 Peptide LD₅₀ LD₉₀₋₁₀₀ MTD MBI 11CN 34 mg/kg 40 mg/kg 20 mg/kgMBI 11B7CN 52 mg/kg >80 mg/kg   30 mg/kg MBI 11F3CN 21 mg/kg 40 mg/kg<20 mg/kg   MBI 11F3CN 52 mg/kg 80 mg/kg 20 mg/kg

The single dose toxicity of a cationic peptide and antibiotic agentcombination is examined in outbred ICR mice. Intraperitoneal injectionof the combination in isotonic saline is carried out at increasing doselevels. The survival of the animals is monitored for 7 days. The numberof animals surviving at each dose level is used to determine the maximumtolerated dose (MTD). In addition, the MTD can be determined afteradministration of the peptide and agent by different routes, atdifferent time points, and in different formulations.

TABLE 32 Peptide # MTD/mg/kg Intraperitoneal injection MBI 10CN >29 MBI11CN >40 MBI 26 >37 MBI 29 24 Intravenous injection MBI 10CN 5.6 MBI11CN 6.1 MBI 26 >18

The single dose toxicity of MBI 10CN and MBI 11CN is examined in outbredICR mice (Table 32). Intraperitoneal injection (groups of 2 mice) of MBI10CN in formulation D showed no toxicity up to 29 mg/kg and under thesame conditions MBI 11CN showed no toxicity up to 40 mg/kg.

Intravenous route. Groups of 6 mice are injected with peptide doses of20, 16, 12, 8, 4 and 0 mg/kg in 100 μl volumes (4 ml/kg). Afteradministration, the mice are observed for a period of 5 days, at whichtime the LD₅₀, LD₉₀₋₁₀₀ and MTD levels are determined. The results fromthe IV toxicity testing of MBI 11CN and three analogues are shown inTable 33. The LD₅₀, LD₉₀₋₁₀₀ and MTD values range from 5.8 to 15 mg/kg,8 to 20 mg/kg and <4 to 12 mg/kg respectively.

TABLE 33 Peptide LD₅₀ LD₉₀₋₁₀₀ MTD MBI 11CN 5.8 mg/kg 8.0 mg/kg  <4mg/kg MBI 11B7CN 7.5 mg/kg 16 mg/kg  4 mg/kg MBI 11E3CN  10 mg/kg 12mg/kg  8 mg/kg MBI 11F4CN  15 mg/kg 20 mg/kg 12 mg/kg

Intravenous injection (groups of 10 mice) of MBI 10CN in formulation Dshowed an MTD of 5.6 mg/kg. Injection of 11 mg/kg gave 40% toxicity and22 mg/kg gave 100% toxicity. Intravenous injection of MBI 11CN informulation C (lyophilized) showed a MTD of 3.0 mg/kg. Injection at 6.1mg/kg gave 10% toxicity and at 12 mg/kg 100% toxicity.

TABLE 34 MTD Peptide Route # Animals Formulation (mg/kg) MBI 10CN ip 2formulation D 29 MBI 11CN ip 2 formulation D 40 MBI 10CN iv 10formulation D 5.6 MBI 11CN iv 10 formulation C 3.0 (lyophilized)

These results are obtained using peptide/buffer solutions that werelyophilized after preparation and reconstituted with water. If thepeptide solution is not lyophilized before injection, but usedimmediately after preparation, an increase in toxicity is seen, and themaximum tolerated dose can decrease by up to four-fold. For example, anintravenous injection of MBI 11CN as a non-lyophilized solution,formulation C1, at 1.5 mg/kg gives 20% toxicity and at 3.0 mg/kg gives100% toxicity. HPLC analyses of the non-lyophilized and lyophilizedformulations indicate that the MBI 11CN forms a complex with Tween 80,and this complexation of the peptide reduces its toxicity in mice.

In addition, mice are multiply injected by an intravenous route with MBI11CN (Table 35). In one representative experiment, peptide administeredin 10 injections of 0.84 mg/kg at 5 minute intervals is not toxic.However, two injections of peptide at 4.1 mg/kg administered with a 10minute interval results in 60% toxicity.

TABLE 35 Dose Level (mg/ # Time Peptide Route Formulation kg) InjectionsInterval Result MBI 11CN iv formulation 0.84 10  5 min no D toxicity MBI11CN iv formulation 4.1 2 10 min 66% D toxicity

Subcutaneous route. The toxicity of MBI 11CN is also determined aftersubcutaneous (SC) administration. For SC toxicity testing, groups of 6mice are injected with peptide doses of 128, 96, 64, 32 and 0 mg/kg in300 μL dose volumes (12 mL/kg). After administration, the mice areobserved for a period of 5 days. None of the animals died at any of thedose levels within the 5 day observation period. Therefore, the LD₅₀,LD₉₀₋₁₀₀ and MTD are all taken to be greater than 128 mg/kg. Micereceiving higher dose levels showed symptoms similar to those seen afterIV injection suggesting that peptide entered the systemic circulation.These symptoms are reversible, disappearing in all mice by the secondday of observations.

The single dose toxicity of MBI 10CN and MBI 11CN in differentformulations is also examined in outbred ICR mice (Table 36).Intraperitoneal injection (groups of 2 mice) of MBI 10CN in formulationD show no toxicity up to 29 mg/kg and under the same conditions MBI 11CNshow no toxicity up to 40 mg/kg.

Intravenous injection (groups of 10 mice) of MBI 10CN in formulation Dshow a maximum tolerated dose (MTD) of 5.6 mg/kg (Table 36). Injectionof 11 mg/kg gave 40% toxicity and 22 mg/kg result in 100% toxicity.Intravenous injection of MBI 11CN in formulation C (lyophilized) show aMTD of 3.0 mg/kg. Injection at 6.1 mg/kg result in 10% toxicity and at12 mg/kg 100% toxicity.

TABLE 36 MTD Peptide Route # Animals Formulation (mg/kg) MBI 10CN ip 2formulation D >29 MBI 11CN ip 2 formulation D >40 MBI 10CN iv 10formulation D 5.6 MBI 11CN iv 10 formulation C 3.0 (lyophilized)

These results are obtained using peptide/buffer solutions that arelyophilized after preparation and reconstituted with water. If thepeptide solution is not lyophilized before injection, but usedimmediately after preparation, an increase in toxicity is seen, and themaximum tolerated dose can decrease by up to four-fold. For example, anintravenous injection of MBI 11CN as a non-lyophilized solution,formulation C1, at 1.5 mg/kg results in 20% toxicity and at 3.0 mg/kggave 100% toxicity. HPLC analyses of the non-lyophilized and lyophilizedformulations indicate that the MBI 11CN forms a complex withpolysorbate, and this complexation of the peptide reduces its toxicityin mice.

In addition, mice are multiply injected by an intravenous route with MBI11CN (Table 37). In one representative experiment, peptide administeredin 10 injections of 0.84 mg/kg at 5 minute intervals is not lethal.However, two injections of peptide at 4.1 mg/kg administered with a 10minute interval results in 60% mortality.

TABLE 37 Formu- Dose # Time Peptide Route lation Level* InjectionsInterval Result MBI 11CN iv formu- 0.84 10  5 min no lation D mortalityMBI 11CN iv formu- 4.1 2 10 min 66% lation D mortality *(mg/kg)

To assess the impact of dosing mice with peptide analogue, a series ofhistopathology investigations can be carried out. Groups of mice areadministered analogue at dose levels that are either at, or below theMTD, or above the MTD, a lethal dose. Multiple injections may be used tomimic possible treatment regimes. Groups of control mice are notinjected or injected with buffer only.

Following injection, mice are sacrificed at specified times and theirorgans immediately placed in a 10% balanced formalin solution. Mice thatdie as a result of the toxic effects of the analogue also have theirorgans preserved immediately. Tissue samples are taken and prepared asstained micro-sections on slides which are then examinedmicroscopically. Damage to tissues is assessed and this information canbe used to develop improved analogues, improved methods ofadministration or improved dosing regimes.

To assess the impact of dosing mice with peptide analogue, a series ofhistopathology investigations are carried out. Groups of two mice areadministered MBI 11CN in formulation D by ip and iv injection. The doselevels are either at or below the MTD or a lethal dose above MTD. Groupsof control mice are uninjected or injected with buffer only. At 0, 70and 150 minutes after injection, the major organs of moribund orsacrificed mice are examined histologically for evidence of toxicity.

Mice Given an IV Injection of MBI 11CN are Identified as Follows:

Control Mouse A: No dose

Control Mouse B: Buffer Dose Only (no peptide)

M70A,B: MBI 11CN, 4 mg/kg, 70 minute observation

M150A,B: MBI 11CN, 4 mg/kg, 150 minute observation

MXA,B: MBI 11CN, 12 mg/kg (lethal dose)

Mice Given an IP Injection of MBI 11CN are Identified as Follows:

Control Mouse A: No dose

Control Mouse B: Buffer Dose Only (no peptide)

M70A,B: MBI 11CN, 40 mg/kg, 70 minute observation

M150A,B: MBI 11CN, 40 mg/kg, 150 minute observation

MXA,B: MBI 11CN, 80 mg/kg (lethal dose)

Following injection, the mice are sacrificed at the times indicatedabove and their organs immediately placed in a 10% balanced formalinsolution. The tissue samples are prepared as stained micro-sections onslides and then examined microscopically.

Mice given a non-lethal dose were always lethargic, with raised fur andevidence of edema and hypertension, but recovered to normal within twohours.

Tissues from these animals indicate that there was some damage to bloodvessels, particularly within the liver and lung at both the observationtimes, but other initial abnormalities returned to normal within the 150minute observation time. It is likely that blood vessel damage is aconsequence of continuous exposure to high circulating peptide levels.

In contrast, mice given a lethal dose had completely normal tissues andorgans, except for the liver and heart of the ip and iv dosed mice,respectively. In general, this damage is identified as disruption of thecells lining the blood vessels. It appears as though the rapid death ofmice is due to this damage, and that the peptide did not penetratebeyond that point. Extensive damage to the hepatic portal veins in theliver and to the coronary arterioles in the heart was observed.

Further evidence points to a cumulative toxic effect, where the maximumdose iv is lethal when repeated after 10 minutes, but not when repeatedafter one hour.

Example 17 In Vivo Efficacy of Cationic Peptides

Cationic peptides are tested for their ability to rescue mice fromlethal bacterial infections. The animal model used is an intraperitoneal(ip) inoculation of mice with 10⁶-10⁸ Gram-positive organisms withsubsequent administration of peptide. The three pathogens investigated,methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus(MRSA), or S. epidermidis are injected ip into mice. For untreated mice,death occurs within 12-18 hours with MSSA and S. epidermis and within6-10 hours with MRSA.

Peptide is administered by two routes, intraperitoneally, at one hourpost-infection, or intravenously, with single or multiple doses given atvarious times pre- and post-infection.

MSSA infection. In a typical protocol, groups of 10 mice are infectedintraperitoneally with a LD₉₀₋₁₀₀ dose (5.2×10⁶ CFU/mouse) of MSSA(Smith, ATCC #19640) injected in brain-heart infusion containing 5%mucin. This strain of S. aureus is not resistant to any commonantibiotics. At 60 minutes post-infection, MBI 10CN or MBI 11CN, informulation D, is injected intraperitoneally at the stated dose levels.An injection of formulation alone serves as a negative control andadministration of ampicillin serves as a positive control. The survivalof the mice is monitored at 1, 2, 3 and 4 hrs post-infection and twicedaily thereafter for a total of 8 days.

As shown in FIG. 17, MBI 10CN is maximally active against MSSA (70-80%survival) at doses of 14.5 to 38.0 mg/kg, although 100% survival is notachieved. Below 14.5 mg/kg, there is clear dose-dependent survival. Atthese lower dose levels, there appears to be an animal-dependentthreshold, as the mice either die by day 2 or survive for the full eightday period. As seen in FIG. 18, MBI 11CN, on the other hand, rescued100% of the mice from MSSA infection at a dose level of 35.7 mg/kg, andwas therefore as effective as ampicillin. There was little or noactivity at any of the lower dose levels, which indicates that a minimumbloodstream peptide level must be achieved during the time that bacteriaare a danger to the host.

As shown above, blood levels of MBI 11CN can be sustained at a level ofgreater than 2 μg/ml for a two hour period inferring that this is higherthan the minimum level.

Additionally, eight variants based on the sequence of MBI 11CN aretested against MSSA using the experimental system described above.Peptides prepared in formulation D are administered at dose levelsranging from 12 to 24 mg/kg and the survival of the infected mice ismonitored for eight days (FIGS. 19-27). The percentage survival at theend of the observation period for each variant is summarized in Table38. As shown in the table, several of the variants showed efficacygreater than or equal to MBI 11CN under these conditions.

TABLE 38 % Survival 24 mg/kg 18 mg/kg 12 mg/kg 100 90 11B1CN, 11F3CN 8070 11E3CN 60 11B7CN 50 11CN 40 11G2CN 30 11B1CN 20 11G4CN 10 11CN,11G2CN 11B7CN, 11B8CN, 11F3CN 0 11A1CN 11A1CN, 11CN, 11G2CN, 11A1CN,11G4CN 11B1CN, 11B7CN, 11B8CN, 11F3CN, 11G4CN

S. epidermidis infection. Peptide analogues generally have lower MICvalues against S. epidermidis in vitro, therefore, lower blood peptidelevels might be more effective against infection.

In a typical protocol, groups of 10 mice are injected intraperitoneallywith an LD₉₀₋₁₀₀ dose (2.0×10⁸ CFU/mouse) of S. epidermidis (ATCC#12228) in brain-heart infusion broth containing 5% mucin. This strainof S. epidermidis is 90% lethal after 5 days. At 15 mins and 60 minspost-infection, various doses of MBI 11CN in formulation D are injectedintravenously via the tail vein. An injection of formulation only servesas the negative control and injection of gentamicin serves as thepositive control; both are injected at 60 minutes post-infection. Thesurvival of the mice is monitored at 1, 2, 3, 4, 6 and 8 hrspost-infection and twice daily thereafter for a total of 8 days.

As shown in FIGS. 28A and 28B, MBI 11CN prolongs the survival of themice. Efficacy is observed at all three dose levels with treatment 15minutes post-infection, however, there is less activity at 30 minutespost-infection and no significant effect at 60 minutes post-infection.Time of administration appears to be important in this model system,with a single injection of 6.1 mg/kg 15 minutes post-infection givingthe best survival rate.

MRSA infection. MRSA infection, while lethal in a short period of time,requires a much higher bacterial load than MSSA. In a typical protocol,groups of 10 mice are injected intraperitoneally with a LD₉₀₋₁₀₀ dose(4.2×10⁷ CFU/mouse) of MRSA (ATCC #33591) in brain-heart infusioncontaining 5% mucin. The treatment protocols are as follows, with thetreatment times relative to the time of infection:

0 mg/kg Formulation D alone (negative control), injected at 0 mins 5mg/kg Three 5.5 mg/kg injections at −5, +55, and +115 mins 1 mg/kg (2hr) Five 1.1 mg/kg injections at −5, +55, +115, +175 and +235 mins 1mg/kg (20 min) Five 1.1 mg/kg injections at −10, −5, 0, +5, and +10 minsVancomycin (positive control) injected at 0 mins

MBI 11CN is injected intravenously in the tail vein in formulation D.Survival of mice is recorded at 1, 2, 3, 4, 6, 8, 10, 12, 20, 24 and 30hrs post-infection and twice daily thereafter for a total of 8 days.There was no change in the number of surviving mice after 24 hrs (FIG.29).

The 1 mg/kg (20 min) treatment protocol, with injections 5 minutes apartcentered on the infection time, delayed the death of the mice to asignificant extent with one survivor remaining at the end of the study.The results presented in Table 39 suggest that a sufficiently high levelof MBI 11CN maintained over a longer time period would increase thenumber of mice surviving. The 5 mg/kg and 1 mg/kg (2 hr) results, wherethere is no improvement in survivability over the negative control,indicates that injections 1 hour apart, even at a higher level, are noteffective against MRSA.

TABLE 39 Time of Observation Percentage of Animals Surviving (Hourspost-infection) No Treatment Treatment 6  50% 70% 8 0 40% 10 0 30% 12 020%

Example 18 Activation of Polysorbate 80 by Ultraviolet Light

A solution of 2% (w/w) polysorbate 80 is prepared in water and 200 mlare placed in a 250 mL crystallizing dish or over suitable container.Containers must have a clear light path. Cover the vessel with a pieceof UV transparent plastic wrap or other UV transparent material. Inaddition, the material should allow the exchange of air but minimizeevaporation.

The solution is irradiated with ultraviolet light using a lamp emittingat 254 nm. Irradiation can also be performed using a lamp emitting at302 nm. The solution should be stirred continuously to maximize the rateof activation. The activation is complete within 72 hours using a lampwith a output of 1800 μW/cm². The reaction is monitored by areversed-phased HPLC assay, which measures the formation of APO-MBI11CN-Tw80 when the light-activated polysorbate is reacted with MBI 11CN.

Some properties of activated polysorbate are determined. Becauseperoxides are a known by-product of exposing ethers to UV light,peroxide formation is examined through the effect of reducing agents onthe activated polysorbate. As seen in FIG. 30A, activated polysorbatereadily reacts with MBI 11CN. Pre-treatment with 2-mercaptoethanol (FIG.30B), a mild reducing agent, eliminates detectable peroxides, but doesnot cause a loss of conjugate forming ability. Treatment with sodiumborohydride (FIG. 30C), eliminates peroxides and eventually eliminatesthe ability of activated polysorbate to modify peptides. Hydrolysis ofthe borohydride in water raises the pH and produces borate as ahydrolysis product. However, neither a pH change nor borate areresponsible.

These data indicate that peroxides are not involved in the modificationof peptides by activated polysorbate. Sodium borohydride should notaffect epoxides or esters in aqueous media, suggesting that the reactivegroup is an aldehyde or ketone. The presence of aldehydes in theactivated polysorbate is confirmed by using a formaldehyde test, whichis specific for aldehydes including aldehydes other than formaldehyde.

Furthermore, activated polysorbate is treated with2,4-dinitrophenylhydrazine (DNPH) in an attempt to capture the reactivespecies. Three DNPH-tagged components are purified and analyzed by massspectroscopy. These components are polysorbate-derived with molecularweights between 1000 and 1400. This indicates that low molecular weightaldehydes, such as formaldehyde or acetaldehyde, are involved.

Example 19 Activation of Polysorbate 80 by Ammonium Persulfate

A 200 mL solution of 2% (w/w) polysorbate 80 is prepared in water. Tothis solution, 200 mg of ammonium persulfate is added while stirring.The reaction is stirred for 1-2 hours with protection from ambientlight. If a solution of less than 0.1% (w/w) ammonium persulfate isused, then exposure to ultraviolet light at 254 nm during this period isused to help complete the reaction. The peroxide level in the reactionis determined using a test kit. Peroxides are reduced by titration with2-mercaptoethanol.

Example 20 Formation of APO-Modified Peptides

APO-modified peptides are prepared either in solid phase or liquidphase. For solid phase preparation, 0.25 ml of 4 mg/ml of MBI 11CN isadded to 0.5 ml of 0.4 M Acetic acid-NaOH pH 4.6 followed by addition of0.25 ml of UV-activated polysorbate. The reaction mix is frozen byplacing it in a −80° C. freezer. After freezing, the reaction mix islyophilized overnight.

For preparing the conjugates in an aqueous phase, a sample of UVactivated polysorbate 80 is first adjusted to a pH of 7.5 by theaddition of 0.1M NaOH. This pH adjusted solution (0.5 ml) is added to1.0 ml of 100 mM sodium carbonate, pH 10.0, followed immediately by theaddition of 0.5 ml of 4 mg/ml of MBI 11CN. The reaction mixture isincubated at ambient temperature for 22 hours. The progress of thereaction is monitored by analysis at various time points using RP-HPLC(FIG. 31). In FIG. 31, peak 2 is unreacted peptide, peak 3 isAPO-modified peptide. Type 1 is the left-most of peak 3 and Type 2 isthe right-most of peak 3.

The table below summarizes data from several experiments. Unlessotherwise noted in the table, the APO-modified peptides are prepared viathe lyophilization method in 200 mM acetic acid-NaOH buffer, pH 4.6.

TABLE 40 COMPLEX SEQUENCE NAME TYPE 1 TYPE 2 ILKKWPWWPWRRKamide 11CN YesLow (SEQ ID NO: 99) Solid phase, pH 2.0 Yes Yes Solid phase, pH 4.6 YesYes Solid phase, pH 5.0 Yes Yes Solid phase, pH 6.0 Yes YesSolid phase, pH 8.3 Trace Trace Solution, pH 2.0 Yes Yes-SlowSolution, pH 10.0 (Ac)₄- 11CN-Y1 No No ILKKWPWWPWRRKamide(SEQ ID NO: 99) ILRRWPWWPWRRKamide 11B1CN Yes Lowered (SEQ ID NO: 41)ILRWPWWPWRRKamide 11B7CN Yes Lowered (SEQ ID NO: 101) ILWPWWPWRRKamide11B8CN Yes Lowered (SEQ ID NO: 66) ILRRWPWWPWRRRamide 11B9CN Yes Trace(SEQ ID NO: 102) ILKKWPWWPWKKKamide 11B10CN Yes Yes (SEQ ID NO: 103)iLKKWPWWPWRRkamide 11E3CN Yes Yes (SEQ ID NO: 99) ILKKWVWWPWRRKamide11F3CN Yes Yes (SEQ ID NO: 59) ILKKWPWWPWKamide 11G13CN Yes Yes(SEQ ID NO: 113) ILKKWPWWPWRamide 11G14CN Yes Trace (SEQ ID NO: 114)

The modification of amino groups is further analyzed by determining thenumber of primary amino groups lost during attachment. The unmodifiedand modified peptides are treated with 2,4,6-trinitrobenzenesulfonicacid (TNBS) (R. L. Lundblad in Techniques in Protein Modification andAnalysis pp. 151-154, 1995).

Briefly, a stock solution of MBI 11CN at 4 mg/ml and an equimolarsolution of APO-modified MBI 11CN are prepared. A 0.225 ml aliquot ofMBI 11CN or APO-modified MBI 11CN is mixed with 0.225 ml of 200 mMsodium phosphate buffer, pH 8.8. A 0.450 ml aliquot of 1% TNBS is addedto each sample, and the reaction is incubated at 37° C. for 30 minutes.The absorbance at 367 nm is measured, and the number of modified primaryamino groups per molecule is calculated using an extinction coefficientof 10,500 M⁻¹ cm⁻¹ for the trinitrophenyl (TNP) derivatives.

The primary amino group content of the parent peptide is then comparedto the corresponding APO-modified peptide. As shown below, the loss of asingle primary amino group occurs during formation of modified peptide.Peptides possessing a 3,4 lysine pair consistently give results that are1 residue lower than expected, which may reflect steric hindrance aftertitration of one member of the doublet.

TABLE 41 TNP/APO- TNP/ modified PEPTIDE SEQUENCE PEPTIDE peptide CHANGEILKKWPWWPWRRKamide 2.71 1.64 1.07 (SEQ ID NO: 99) ILRRWPWWPWRRKamide1.82 0.72 1.10 (SEQ ID NO: 41) IIKKWPWWPWRRkamide 2.69 1.61 1.08(SEQ ID NO: 99) ILKKWVWWPWRRKamide 2.62 1.56 1.06 (SEQ ID NO: 59)

Stability of APO-Modified Peptide Analogues

APO-modified peptides demonstrate a high degree of stability underconditions that promote the dissociation of ionic or hydrophobiccomplexes. APO-modified peptide in formulation D is prepared as 800μg/ml solutions in water, 0.9% saline, 8M urea, 8M guanidine-HCl, 67%1-propanol, 1M HCl and 1M NaOH and incubated for 1 hour at roomtemperature. Samples are analyzed for the presence of free peptide usingreversed phase HPLC and the following chromatographic conditions:

Solvent A: 0.1% trifluoroacetic acid (TFA) in water

Solvent B: 0.1% TFA 195% acetonitrile in water

Media: POROS R2-20 (polystyrene divinylbenzene)

Elution: 0% B for 5 column volumes

-   -   0-25% B in 3 column volumes    -   25% B for 10 column volumes    -   25-95% B in 3 column volumes    -   95% B for 10 column volumes

Under these conditions, free peptide elutes exclusively during the 25% Bstep and formulation-peptide complex during the 95% B step. None of thedissociating conditions mentioned above, with the exception of 1M NaOHin which some degradation is observed, are successful in liberating freepeptide from APO-modified peptide. Additional studies are carried outwith incubation at 55° C. or 85° C. for one hour. APO-modified peptideis equally stable at 55° C. and is only slightly less stable at 85° C.Some acid hydrolysis, indicated by the presence of novel peaks in theHPLC chromatogram, is observed with the 1M HCl sample incubated at 85°C. for one hour.

Example 21 Purification of APO-Modified MBI 11CN

A large scale preparation of APO-modified MBI 11CN is purified.Approximately 400 mg of MBI 11CN is APO-modified and dissolved in 20 mlof water. Unreacted MBI 11CN is removed by RP-HPLC. The solvent is thenevaporated from the APO-modified MBI 11CN pool, and the residue isdissolved in 10 ml methylene chloride. The modified peptide is thenprecipitated with 10 ml diethyl ether. After 5 min at ambienttemperature, the precipitate is collected by centrifugation at 5000×gfor 10 minutes. The pellet is washed with 5 ml of diethyl ether andagain collected by centrifugation at 5000×g for 10 minutes. Thesupernatants are pooled for analysis of unreacted polysorbateby-products. The precipitate is dissolved in 6 ml of water and thenflushed with nitrogen by bubbling for 30 minutes to remove residualether. The total yield from the starting MBI 11CN was 43%.

The crude APO-MBI29-Tw80 prepared from 200 mg of MBI 29 is suspended in40 mL of methylene chloride and sonicated for 5 minutes to disperselarge particles. The suspension is centrifuged in appropriate containers(Corning glass) at 3000-4000×g for 15 minutes at 10° C. to sedimentinsoluble material. The supernatant is decanted and saved.

The sediment is extracted twice more by adding 40 mL portions methylenechloride to the sediment and repeating the sonication/centrifugationstep. The supernatants from the three extractions are pooled andconcentrated 8-10 fold using a rotary evaporator. The solution istransferred to centrifuge tubes and 3 volumes of diethyl ether areadded. The mixture is incubated for 15 minutes, then centrifuged at3000-4000×g for 15 minutes at 10° C. to sediment the product. Thesupernatant is decanted and discarded. The residual ether may be removedwith a stream of nitrogen.

Example 22 Biological Assays to Measure APO-Cationic Peptide Activity

All biological assays that compare APO-modified peptides with unmodifiedpeptides are performed on an equimolar ratio. The concentration ofAPO-modified peptides can be determined by spectrophotometricmeasurement, which is used to normalize concentrations for biologicalassays. For example, a 1 mg/ml APO-modified MBI 11CN solution containsthe same amount of peptide as a 1 mg/ml MBI 11CN solution, thus allowingdirect comparison of toxicity and efficacy data.

APO-modified peptides are at least as potent as the parent peptides inin vitro assays performed as described herein. MIC values against grampositive bacteria are presented for several APO-modified peptides andcompared with the values obtained using the parent peptides (Table 5).The results indicate that the modified peptides are at least as potentin vitro as the parent peptides and may be more potent than the parentpeptides against E. faecalis strains.

The agarose dilution assay measures antimicrobial activity of peptidesand peptide analogues, which is expressed as the minimum inhibitoryconcentration (MIC) of the peptides. This assay is performed asdescribed above. Representative MICs for various modified and unmodifiedcationic peptides are shown in the Table below.

TABLE 42 MIC (μg/mL) APO- Organism Organism # APO-Peptide PeptidePeptide A. calcoaceticus AC002 MBI11CN-Tw80 4 4 A. calcoaceticus AC002MBI11B1CN-Tw80 4 2 A. calcoaceticus AC002 MBI11B7CN-Tw80 4 2 A.calcoaceticus AC002 MBI11B7CN-Tx114r 2 2 A. calcoaceticus AC002MBI11B7CN-F12-Tx114r 1 1 A. calcoaceticus AC002 MBI11E3CN-Tw80 2 1 A.calcoaceticus AC002 MBI11F3CN-Tw80 8 2 A. calcoaceticus AC002MBI11F4CN-Tw80 4 4 A. calcoaceticus AC002 MBI29-Tw80 4 1 E. cloacaeECL007 MBI11CN-Tw80 >128 >128 E. cloacae ECL007 MBI11B1CN-Tw80 128 >128E. cloacae ECL007 MBI11B7CN-Tw80 >128 128 E. cloacae ECL007MBI11B7CN-Tx114r 128 128 E. cloacae ECL007MBI11B7CN-F12-Tx114r >128 >128 E. cloacae ECL007 MBI11E3CN-Tw80 128 >128E. cloacae ECL007 MBI11F3CN-Tw80 128 >128 E. cloacae ECL007MBI11F4CN-Tw80 64 32 E. cloacae ECL007 MBI29-Tw80 32 >64 E. coli ECO005MBI11CN-Tw80 16 8 E. coli ECO005 MBI11B1CN-Tw80 8 8 E. coli ECO005MBI11B7CN-Tw80 16 4 E. coli ECO005 MBI11B7CN-Tx114r 16 4 E. coli ECO005MBI11B7CN-F12-Tx114r 32 16 E. coli ECO005 MBI11E3CN-Tw80 8 4 E. coliECO005 MBI11F3CN-Tw80 128 16 E. coli ECO005 MBI11F4CN-Tw80 8 8 E. coliECO005 MBI29-Tw80 16 4 E. faecalis EFS001 MBI11CN-Tw80 8 32 E. faecalisEFS001 MBI11B1CN-Tw80 4 32 E. faecalis EFS001 MBI11B7CN-Tw80 8 8 E.faecalis EFS001 MBI11B7CN-Tx114r 0.5 0.5 E. faecalis EFS001MBI11B7CN-F12-Tx114r 0.5 0.5 E. faecalis EFS001 MBI11E3CN-Tw80 4 8 E.faecalis EFS001 MBI11F3CN-Tw80 8 32 E. faecalis EFS001 MBI29-Tw80 0.50.5 E. faecalis EFS004 MBI11CN-Tw80 4 8 E. faecalis EFS004MBI11B1CN-Tw80 4 8 E. faecalis EFS004 MBI11B7CN-Tw80 8 8 E. faecalisEFS004 MBI11E3CN-Tw80 4 2 E. faecalis EFS004 MBI11F3CN-Tw80 4 16 E.faecalis EFS008 MBI11CN-Tw80 1 16 E. faecalis EFS008 MBI11B1CN-Tw80 1 2E. faecalis EFS008 MBI11B7CN-Tw80 1 2 E. faecalis EFS008MBI11B7CN-Tx114r 2 4 E. faecalis EFS008 MBI11B7CN-F12-Tx114r 2 2 E.faecalis EFS008 MBI11E3CN-Tw80 1 2 E. faecalis EFS008 MBI11F3CN-Tw80 416 E. faecalis EFS008 MBI11F4CN-Tw80 2 2 E. faecalis EFS008 MBI29-Tw80 20.5 K. pneumoniae KP001 MBI11CN-Tw80 8 16 K. pneumoniae KP001MBI11B1CN-Tw80 8 8 K. pneumoniae KP001 MBI11B7CN-Tw80 8 4 K. pneumoniaeKP001 MBI11B7CN-Tx114r 8 8 K. pneumoniae KP001 MBI11B7CN-F12-Tx114r 3216 K. pneumoniae KP001 MBI11E3CN-Tw80 4 8 K. pneumoniae KP001MBI11F3CN-Tw80 128 64 K. pneumoniae KP001 MBI11F4CN-Tw80 8 4 K.pneumoniae KP001 MBI29-Tw80 16 2 P. aeruginosa PA004 MBI11CN-Tw80 >128128 P. aeruginosa PA004 MBI11B1CN-Tw80 128 64 P. aeruginosa PA004MBI11B7CN-Tw80 128 128 P. aeruginosa PA004 MBI11B7CN-Tx114r 128 128 P.aeruginosa PA004 MBI11B7CN-F12-Tx114r >128 >128 P. aeruginosa PA004MBI11E3CN-Tw80 64 32 P. aeruginosa PA004 MBI11F3CN-Tw80 128 128 P.aeruginosa PA004 MBI11F4CN-Tw80 128 32 P. aeruginosa PA004MBI29-Tw80 >64 16 S. aureus SA010 MBI11B1CN 4 1 S. aureus SA010MBI11B7CN 4 1 S. aureus SA010 MBI11CN 4 2 S. aureus SA010 MBI11E3CN 2 1S. aureus SA010 MBI11F3CN 4 2 S. aureus SA011 MBI11CN-Tw80 16 8 S.aureus SA011 MBI11B1CN-Tw80 16 4 S. aureus SA011 MBI11B7CN-Tw80 16 4 S.aureus SA011 MBI11E3CN-Tw80 16 4 S. aureus SA011 MBI11F3CN-Tw80 16 8 S.aureus SA014 MBI11CN-Tw80 2 1 S. aureus SA014 MBI11B1CN-Tw80 2 1 S.aureus SA014 MBI11B7CN-Tw80 1 2 S. aureus SA014 MBI11B7CN-Tx114r 2 1 S.aureus SA014 MBI11B7CN-F12-Tx114r 2 2 S. aureus SA014 MBI11E3CN-Tw80 1 1S. aureus SA014 MBI11F3CN-Tw80 8 8 S. aureus SA014 MBI11F4CN-Tw80 2 2 S.aureus SA014 MBI29-Tw80 2 1 S. aureus SA018 MBI11CN-Tw80 64 64 S. aureusSA018 MBI11B1CN-Tw80 32 16 S. aureus SA018 MBI11B7CN-Tw80 32 16 S.aureus SA018 MBI11E3CN-Tw80 32 16 S. aureus SA018 MBI11F3CN-Tw80 64 16S. aureus SA025 MBI11CN-Tw80 2 4 S. aureus SA025 MBI11B1CN-Tw80 4 1 S.aureus SA025 MBI11B7CN-Tw80 2 1 S. aureus SA025 MBI11E3CN-Tw80 2 1 S.aureus SA025 MBI11F3CN-Tw80 4 2 S. aureus SA093 MBI11CN-Tw80 2 2 S.aureus SA093 MBI11B1CN-Tw80 2 1 S. aureus SA093 MBI11B7CN-Tw80 2 1 S.aureus SA093 MBI11B7CN-Tx114r 1 1 S. aureus SA093 MBI11B7CN-F12-Tx114r 11 S. aureus SA093 MBI11E3CN-Tw80 2 1 S. aureus SA093 MBI11F3CN-Tw80 2 1S. aureus SA093 MBI29-Tw80 1 0.5 S. epidermidis SE010 MBI11B7CN-Tx114r 42 S. epidermidis SE010 MBI11B7CN-F12-Tx114r 4 8 S. epidermidis SE010MBI29-Tw80 >64 4 S. maltophilia SMA002 MBI11CN-Tw80 32 >128 S.maltophilia SMA002 MBI11B1CN-Tw80 32 32 S. maltophilia SMA002MBI11B7CN-Tw80 64 16 S. maltophilia SMA002 MBI11B7CN-Tx114r 32 16 S.maltophilia SMA002 MBI11B7CN-F12-Tx114r 64 64 S. maltophilia SMA002MBI11E3CN-Tw80 128 64 S. maltophilia SMA002 MBI11F3CN-Tw80 128 64 S.maltophilia SMA002 MBI11F4CN-Tw80 32 16 S. maltophilia SMA002 MBI29-Tw808 2 S. marcescens SMS003 MBI11CN-Tw80 >128 >128 S. marcescens SMS003MBI11B1CN-Tw80 >128 >128 S. marcescens SMS003 MBI11B7CN-Tw80 >128 >128S. marcescens SMS003 MBI11B7CN-Tx114r >128 >128 S. marcescens SMS003MBI11B7CN-F12-Tx114r >128 >128 S. marcescens SMS003 MBI11E3CN-Tw80128 >128 S. marcescens SMS003 MBI11F3CN-Tw80 128 >128 S. marcescensSMS003 MBI11F4CN-Tw80 >128 >128 S. marcescens SMS003 MBI29-Tw80 >64 >128

Toxicities of APO-modified MBI 11CN and unmodified MBI 11CN are examinedin Swiss CD-1 mice. Groups of 6 mice are injected iv with single dosesof 0.1 ml peptide in 0.9% saline. The dose levels used are 0, 3, 5, 8,10, and 13 mg/kg. Mice are monitored at 1, 3, and 6 hrs post-injectionfor the first day, then twice daily for 4 days. The survival data forMBI 11CN mice are presented in Table 43. For APO-modified MBI 11CN, 100%of the mice survived at all doses, including the maximal dose of 13mg/kg.

TABLE 43 Peptide Cumu- Cumulative administered No. Dead/ lative No. No.% (mg/kg) Total Dead Surviving Dead/Total Dead 13 6/6 18 0 18/18 100 106/6 12 0 12/12 100 8 6/6 6 0 6/6 100 5 0/6 0 6 0/6 0 3 0/6 0 12 0/12 0 00/6 0 18 0/18 0

As summarized below, the LD₅₀ for MBI 11CN is 7 mg/kg (Table 7), withall subjects dying at a dose of 8 mg/ml. The highest dose of MBI 11CNgiving 100% survival was 5 mg/kg. The data show that APO-modifiedpeptides are significantly less toxic than the parent peptides.

TABLE 44 Test Peptide LD₅₀ LD₉₀₋₁₀₀ MTD MBI 11CN 7 mg/kg 8 mg/kg 5 mg/kgAPO-MBI-11CN >13 mg/kg*  >13 mg/kg*  >13 mg/kg*  *could not becalculated with available data.

In addition, APO-peptides and parent peptides are tested against a panelof cancer cell lines. Cell death is measured using the Cytotox (Promega)assay kit which measures the release of lactate dehydrogenase. As shownbelow the modified peptides had increased activity over the parentpeptides.

TABLE 45 CELL LINE, LC₅₀, μg/mL ± S.E. MCF- Peptide PBL HUVEC H460 K562DoHH-2 P388 P388ADR MCF-7 7ADR 11CN    57 >190 200 — — 30 25  11.8 ± 9 17 ± 1 11CN-Tw80 6 ± 6 16 ± 4 16 ± 4 — — 1.9 ± 5 3.5 ± 2 11 —11A3CN >500 >500 >500 >500    >500      >300 >300    — — 11A3CN-Tw8012.7 ± 15   17 ± 9 15 ± 4 6  3.3 ± 0.05 5.6 ± 2 6.6 ± 3 28 13 11B7CN 24± 10  90 ± 23  26 ± 25 34 ± 25 16.5 ± 3   13.8 — >700    — 11B7CN-Tw803.8 ± 1   12.8 ± 8   >100 4.7 ± 3   3.3 ± 1   5.1 — 12 — 11E3CN 22 ± 11117 ± 7  18 9 3.6 13.9 ± 3  7.9 ± 3  5.6 ± 2 5.3 ± 1 11E3CN-Tw80 4.5 ±2   12.8 ± 2   8.2 ± 4 4.9 ± 3   3.5 ± 0.7 5.9 ± 3 8.4 ± 1  8.1 ± 5 7.6± 2 21A11 30 ± 15  184 ± 100 48 56 ± 33 9.8 ± 0.3 — — — — 21A11-Tw80 4.5± 4     17 ± 9.9 21 4.3 ± 2   4.7 ± 0.6   8.1 ± 3.4 9 18 — 29 12 ± 10   10 12.6 ± 10 1 2.1 ± 0.5   1.4 ± 0.5    2 ± 0.2   4 ± 2 3.2 ± 129-Tw80 8.7 ± 6   9.3 ± 2  1.7 2.1 ± 0.5   4 ± 0.5   7.6 ± 2.4 7.6 ± 215.5 ± 6 9.1 ± 5 PBL, peripheral blood lymphocytes; HUVEC, humanumbilical vein endothelial cells; H460, non-small lung tumor; K562,chronic myelogenous leukemia; DoHH-2, B-cell cell lymphoma; P388,lymphocytic leukemia; P388ADR, lymphocytic leukemia, multidrugresistant; MCF-7, breast carcinoma; MCF-7ADR, breast carcinoma,multidrug resistant.

It will be appreciated that, although specific embodiments of theinvention have been described herein for purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. Accordingly, the invention is not limited exceptas by the appended claims.

1-3. (canceled)
 4. A method of treating or preventing a microbialinfection, comprising administering to a patient a therapeuticallyeffective amount of a composition comprising at least one indolicidinanalogue of up to 35 amino acids that comprises one of the followingsequences: (SEQ ID NO: 25) Lys Arg Arg Trp Pro Trp Trp Pro Trp Lys Lys Leu Ile; (SEQ ID NO: 23) Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg  Arg Lys; (SEQ ID NO: 46) Lys Arg Arg Trp Pro Trp Trp Pro Trp Arg  Leu Ile; (SEQ ID NO: 47) Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg  Arg Lys Ile Met Ile Leu Lys Lys Ala Gly  Ser; (SEQ ID NO: 24)Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys Met Ile Leu Lys Lys Ala Gly Ser; (SEQ ID NO: 48)Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg  Arg Lys Asp Met Ile Leu Lys Lys Ala Gly  Ser; (SEQ ID NO: 26)Trp Arg Ile Trp Lys Pro Lys Trp Arg Leu  Pro Lys Trp; (SEQ ID NO: 59)Ile Leu Lys Lys Trp Val Trp Trp Pro Trp   Arg Arg Lys; (SEQ ID NO: 27)Ile Leu Arg Trp Val Trp Trp Val Trp Arg   Arg Lys; or (SEQ ID NO: 60)Lys Arg Arg Trp Val Trp Trp Val Trp Arg   Leu Ile.


5. The method of claim 4, wherein the indolicidin analogue comprises thesequence of Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys (SEQ IDNO:23).
 6. The method of claim 4, wherein the amino acid sequence of theindolicidin analogue consists of Ile Leu Arg Trp Pro Trp Trp Pro Trp ArgArg Lys (SEQ ID NO:23).
 7. The method of claim 4, wherein the microbialinfection is due to a bacterium and wherein the composition isadministered ex vivo.
 8. The method of claim 7, wherein the compositionis administered to the skin of the patient.
 9. The method of claim 4,wherein the microbial infection is associated with nasal colonization.10. The method of claim 9, wherein the composition is administered byaerosolization or spray.
 11. The method of claim 4, wherein themicrobial infection is a burn-related infection.
 12. The method of claim4, wherein the microbial infection is a surgical wound infection. 13.The method of claim 4, wherein the composition is a gel.
 14. The methodof claim 6, wherein the microbial infection is associated with nasalcolonization.
 15. The method of claim 14, wherein the composition isadministered by aerosolization or spray.
 16. The method of claim 6,wherein the microbial infection is a burn-related infection.
 17. Themethod of claim 6, wherein the microbial infection is a surgical woundinfection.
 18. The method of claim 6, wherein the composition is a gel.19. The method of claim 4, wherein the composition further comprises anantibiotic.
 20. The method of claim 4, further comprising the step ofadministering an antibiotic.
 21. The method of claim 20, wherein theantibiotic is minocycline, rifampin, cefazolin, vancomycin, teicoplanin,or a beta lactam.
 22. The method of claim 4, wherein the microbialinfection is due to a bacterium.
 23. The method of claim 22, wherein thebacterium is antibiotic resistant.
 24. The method of claim 23, whereinthe antibiotic resistant bacteria is resistant to at least one of apenicillin, a cephalosporin, a fluoroquinolone, a macrolide, or anaminoglycoside.
 25. The method of claim 23, wherein the antibioticresistant bacteria is resistant to at least one of a tetracycline, aquinolone, a glycopeptide, a monobactam, a carbacephem, a carbapenem, ora cephamycin.
 26. The method of claim 23, wherein the antibioticresistant bacteria is resistant to at least one of methicillin,vancomycin, teicoplanin, ciprofloxacin, clindamycin, ampicillin,erythromycin, amikacin, ceftriaxone, gentamicin, mupirocin,piperacillin, or tobramycin.
 27. A method of inhibiting or preventinggrowth of a bacterium on a surface of an object, comprising coating thesurface with a therapeutically effective amount of a compositioncomprising at least one indolicidin analogue of up to 35 amino acidsthat comprises one of the following sequences: (SEQ ID NO: 25)Lys Arg Arg Trp Pro Trp Trp Pro Trp Lys   Lys Leu Ile; (SEQ ID NO: 23)Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg   Arg Lys; (SEQ ID NO: 46)Lys Arg Arg Trp Pro Trp Trp Pro Trp Arg   Leu Ile; (SEQ ID NO: 47)Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg  Arg Lys Ile Met Ile Leu Lys Lys Ala Gly  Ser; (SEQ ID NO: 24)Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys Met Ile Leu Lys Lys Ala Gly Ser; (SEQ ID NO: 48)Ile Leu Arg Trp Pro Trp Trp Pro Trp ArgArg Lys Asp Met Ile Leu Lys Lys Ala Gly  Ser; (SEQ ID NO: 26)Trp Arg Ile Trp Lys Pro Lys Trp Arg Leu   Pro Lys Trp; (SEQ ID NO: 59)Ile Leu Lys Lys Tip Val Trp Trp Pro Trp   Arg Arg Lys; (SEQ ID NO: 27)Ile Leu Arg Trp Val Trp Trp Val Trp Arg  Arg Lys; or (SEQ ID NO: 60)Lys Arg Arg Trp Val Trp Trp Val Trp Arg   Leu Ile.


28. The method of claim 27, wherein the indolicidin analogue comprisesthe sequence of Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys (SEQ IDNO:23).
 29. The method of claim 27, wherein the amino acid sequence ofthe indolicidin analogue consists of Ile Leu Arg Trp Pro Trp Trp Pro TrpArg Arg Lys (SEQ ID NO:23).
 30. The method of claim 27, furthercomprising the step of contacting the surface of the object with anantibiotic.
 31. The method of claim 30, wherein the antibiotic isminocycline, rifampin, cefazolin, vancomycin, teicoplanin, or a betalactam.
 32. The method of claim 27, wherein the bacterium is antibioticresistant.
 33. The method of claim 32, wherein the antibiotic resistantbacteria is resistant to at least one of a penicillin, a cephalosporin,a fluoroquinolone, a macrolied, an aminoglycoside, a tetracycline, aquinolone, a glycopeptide, a monobactam, a carbacephem, a carbapenem, ora cephamycin.
 34. The method of claim 27, wherein the object is a stent,tube, probe, cannula, catheter, synthetic vascular graft, bloodmonitoring device, artificial heart valve, or needle.
 35. The method ofclaim 27, wherein the object is a tube.
 36. The method of claim 27,wherein the object is a medical device.
 37. The method of claim 36,wherein the medical device is a catheter.
 38. The method of claim 36,wherein the medical device is an intravascular device.
 39. The method ofclaim 27, wherein the bacteria is a Gram-negative bacteria selected fromthe group consisting of Enterobacter sp., E. coli, Klebsiella sp., andAcinetobacter sp.
 40. The method of claim 27, wherein the bacteria is aGram-positive bacteria selected from the group consisting of S. aureus,coagulase negative staphylococci, enterococci, S. epidermidis, S.pneumoniae, and Viridans Streptococci.
 41. The method of claim 27,wherein the composition is a gel.